Effects of Prenatal Bisphenol A Exposure on Adrenal Gland ...

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Effects of Prenatal Bisphenol A Exposure on Adrenal Gland Effects of Prenatal Bisphenol A Exposure on Adrenal Gland

Development and Steroidogenic Function Development and Steroidogenic Function

Samantha Medwid The University of Western Ontario

Supervisor

Yang, Kaiping

The University of Western Ontario

Graduate Program in Physiology and Pharmacology

A thesis submitted in partial fulfillment of the requirements for the degree in Doctor of

Philosophy

© Samantha Medwid 2017

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Abstract

Developmental exposure to bisphenol A (BPA), a ubiquitous endocrine disrupting

chemical, is associated with organ dysfunction and diseases in adulthood. However, little

is known about its effects on the adrenal glands. Therefore, this thesis addresses this

important question using both in vivo and in vitro approaches. BPA at environmentally

relevant doses was administrated via diet to pregnant mice from embryonic day 7.5 to

birth, following which mice were switched to a standard chow. At two months

postnatally, adrenal glands and blood samples were collected from adult mouse offspring

for structural and functional analysis. I found that: (a) BPA increased adrenal gland

weight as well as plasma corticosterone levels; (b) BPA did not alter plasma levels of

ACTH; and (c) BPA stimulated expression of the two key steroidogenic factors,

steroidogenic acute regulatory protein (StAR) and cyp11A1 in female but not male

offspring. To determine the molecular mechanisms underlying the BPA-induced StAR

expression, I used human fetal adrenal cortical H295A cells as an in vitro model system,

and showed that BPA increased StAR protein expression likely through an estrogen

receptor (ER)-mediated mechanism independent of StAR gene transcription, translation

and protein half-life. I then investigated the molecular mechanisms underlying the BPA-

induced increase in adrenal gland weight using the same in vitro model system. I

demonstrated that (a) BPA increased cell number and protein levels of the three universal

markers of proliferation (proliferating cell nuclear antigen (PCNA), cyclin D1 and D2, as

well as sonic hedgehog (shh) and its key transcriptional regulator Gli1; (b) cyclopamine,

a shh pathway inhibitor, blocked these stimulatory effects of BPA on cell proliferation;

(c) BPA increased the nuclear translocation of ERβ; and (d) the ERb-specific agonist

DPN mimicked while the ERb antagonist PHTPP abrogated the stimulatory effects of

BPA on cell proliferation, and prevented BPA-induced activation of the shh signaling.

Taken together, these findings demonstrate that developmental exposure to BPA

adversely affects adrenal gland development and steroidogenic function in adult mouse

offspring. Furthermore, they reveal novel molecular signaling mechanisms of BPA

actions in regulating adrenal steroidogenic function and adrenal cortical cell proliferation.

ii

Keywords

Bisphenol A, endocrine disruptor, adrenal gland, steroidogenesis, steroidogenic acute regulatory protein, fetal development, estrogen receptor, sonic hedgehog

iii

Co-Authorship Statement

Chapter 3:

Medwid S, Guan H, and Yang K (2016) Prenatal exposure to bisphenol A disrupts adrenal steroidogenesis in adult mouse offspring. Environ Toxicol Pharmacol. 43: 203-8

SM, GH, and KY designed the experiments. SM was responsible for animal care, blood and tissue collection and conducted the experiments. SM, GH and KY analyzed and interpreted the data. SM and KY wrote the manuscript. All authors approved the final version of the manuscript.

Chapter 4:

Medwid S, Guan H, Yang K (2017) Bisphenol A induces steroidogenic acute regulatory protein (StAR) expression via an unknown mechanism independent of transcription, translation and protein half-life in human adrenal cortical cells. Submitted Steroids.

SM, GH and KY designed experiments. SM conducted all experiments. SM, GH and KY analyzed and interpreted the data. SM and KY wrote the manuscript. All authors approved the final version of the manuscript.

Chapter 5:

Medwid S, Guan H, Yang K (2017) Bisphenol A stimulates adrenal cortical cell proliferation via ERβ-mediated activation of the sonic hedgehog signaling pathway. Submitted Journal of Steroid Biochemistry and Molecular Biology.

SM, GH and KY designed experiments. SM conducted all experiments. SM, GH and KY analyzed and interpreted the data. SM and KY wrote the manuscript. All authors approved the final version of the manuscript.

iv

Acknowledgments I would first like to express my deepest appreciation to my advisor, Dr. Kaiping Yang,

whose expertise, leadership, and encouragement has been invaluable to me throughout

this whole experience. I appreciate all your guidance as it has helped me to mature as a

researcher and an individual. It is under your direction that I have discovered the

confidence in my capacity to grow, both academically and personally. I will always

remember the lessons I have learned from you as I begin the next chapter of my life.

I would like to thank my advisory committee members, Dr. Dean Betts, Dr. Andy

Babwah, Dr. Dan Hardy, and Dr. Rommel Tirona. Your guidance, advice and support

through my graduate studies is deeply appreciated.

I would like to express my heartfelt appreciation of Dr. Haiyan Guan. Thank you for

sharing all your technical expertise and support with me over the years but above all,

thank you for your amazing friendship. Special thanks to all the members of the Yang

lab, including Dr. Maria Cernea, Dr. Ayten Hijazi, Bianca DeBenedictis, Colleen

Westerman and Maria Abou Taka. Thank you, to all the other CHRI trainees for all the

many coffee breaks, especially, Amanda Oakie, Jason Peart, Kurt Berger, and Phyo Win.

Throughout the years, each has had an impact in my life in some way or another. The

friendships we have created over the years will always remain in my life and I will

remember all the great times we shared together.

Lastly, I would like to thank my parents and my partner. Thank you to my mom and dad

for their constant encouragement and endless love, which kept me motivated during

difficult times. You have taught me that I can achieve anything I put my mind too. Lastly,

my partner and best friend, Martin, thank you for always supporting and believing in me.

Without the support and encouragement of my family, this process would have been

much more difficult. Thank you!

v

Table of Contents

Abstract ................................................................................................................................ i

Co-Authorship Statement ................................................................................................... iii

Acknowledgments .............................................................................................................. iv

Table of Contents ................................................................................................................ v

List of Tables ...................................................................................................................... x

List of Figures .................................................................................................................... xi

List of Appendices ........................................................................................................... xiii

List of Abbreviations ....................................................................................................... xiv

1 INTRODUCTION ......................................................................................................... 1

1.1 Bisphenol A ............................................................................................................ 2

1.1.1 Mechanism of Action .................................................................................. 3

1.1.2 Dose Response and Low Dose Effects ....................................................... 6

1.1.3 Pharmacokinetics ........................................................................................ 7

1.1.4 Human Exposure to BPA .......................................................................... 11

1.2 The Adrenal Gland ................................................................................................ 13

1.2.1 The HPA Axis ........................................................................................... 16

1.2.2 Physiological Function.............................................................................. 18

1.2.3 Adrenal Development ............................................................................... 21

1.2.4 Adrenal Remodeling and Growth ............................................................. 26

1.2.5 Sex Specificity in Adrenal Glands ............................................................ 34

1.3 Adrenal Steroidogenesis ....................................................................................... 34

1.3.1 Steroidogenic Pathway .............................................................................. 34

1.3.2 Steroidogenic Acute Regulatory Protein .................................................. 39

1.4 Effects of BPA on Steroidogenesis ....................................................................... 44

vi

1.4.1 Male Reproductive Steroidogenesis ......................................................... 44

1.4.2 Female Reproductive Steroidogenesis ...................................................... 44

1.4.3 Adrenal Steroidogenesis ........................................................................... 49

1.5 Rationale ............................................................................................................... 52

1.6 Hypothesis............................................................................................................. 52

1.7 Objectives ............................................................................................................. 52

1.8 References ............................................................................................................. 53

2 PRENATAL EXPOSURE TO BISPHENOL A DISRUPTS STEROIDOGENESIS IN ADULT MOUSE OFFSPRING1 ............................................................................ 72

2.1 Introduction ........................................................................................................... 73

2.2 Materials and Methods .......................................................................................... 74

2.2.1 Animal Experiments ................................................................................. 74

2.2.2 Western Blot Analysis .............................................................................. 74

2.2.3 Hormone Assays ....................................................................................... 77

2.2.4 Statistical Analysis .................................................................................... 77

2.3 Results ................................................................................................................... 77

2.3.1 Effects of prenatal BPA exposure on adrenal gland weight ..................... 77

2.3.2 Effects of prenatal BPA exposure on basal plasma corticosterone and ACTH levels ............................................................................................. 79

2.3.3 Effects of prenatal BPA exposure on perilipin protein levels ................... 81

2.3.4 Effects of prenatal BPA exposure on StAR and cyp11A1 protein levels . 83

2.3.5 Effects of prenatal BPA exposure on SF-1 protein levels ........................ 85

2.4 Discussion ............................................................................................................. 87

2.5 Conclusion ............................................................................................................ 91

2.6 References ............................................................................................................. 91

3 BISPHENOL A INDUCES STEROIDOGENIC ACUTE REGULATORY PROTEIN (StAR) EXPRESSION VIA AN UNKNOWN MECHANISM

vii

INDEPENDENT OF TRANSCRIPTION, TRANSLATION AND PROTEIN HALF-LIFE IN HUMAN ADRENAL CORTICAL CELLS1 .................................... 96

3.1 Introduction ........................................................................................................... 97

3.2 Methods................................................................................................................. 98

3.2.1 Reagents .................................................................................................... 98

3.2.2 Cell Culture ............................................................................................... 98

3.2.3 Western Blot Analysis .............................................................................. 98

3.2.4 Real time quantitative RT-PCR .............................................................. 101

3.2.5 Statistical Analysis .................................................................................. 101

3.3 Results ................................................................................................................. 102

3.3.1 Concentration-dependent effects of BPA on StAR protein expression .. 102

3.3.2 Effects of BPA on selected key steroidogenic enzymes ......................... 104

3.3.3 Effects of BPA on ERα and β protein expression ................................... 106

3.3.4 Effects of ER agonists and antagonist on StAR protein expression ....... 108

3.3.5 Effects of BPA on key StAR transcription factors and StAR mRNA levels ....................................................................................................... 110

3.3.6 Effects of BPA on key StAR translation protein and StAR pre-protein . 112

3.3.7 Effects of BPA on half-life of StAR protein ........................................... 114

3.4 Discussion ........................................................................................................... 116

3.5 References ........................................................................................................... 120

4 BISPHENOL A STIMULATES ADRENAL CELL PROLIFERATION THROUGH ERb-MEDIATED ACTIVATION OF THE SONIC HEDGEHOG SIGNALING PATHWAY1 ........................................................................................ 127

4.1 Introduction ......................................................................................................... 128

4.2 Methods............................................................................................................... 129

4.2.1 Reagents .................................................................................................. 129

4.2.2 Cell Culture ............................................................................................. 129

4.2.3 Western Blot Analysis ............................................................................ 130

viii

4.2.4 Cell Number Assessment ........................................................................ 132

4.2.5 Real-time quantitative RT-PCR .............................................................. 132

4.2.6 Statistical Analysis .................................................................................. 134

4.3 Results ................................................................................................................. 134

4.3.1 Time- and concentration-dependent effects of BPA on cell proliferation. ............................................................................................ 134

4.3.2 Effects of BPA on the expression of key cell proliferation factors. ....... 136

4.3.3 Effects of BPA on selected components of the shh signaling pathway. . 138

4.3.4 Effects of BPA on activity of the shh signaling pathway. ...................... 140

4.3.5 Effects of shh pathway inhibition on BPA-induced cell proliferation markers. ................................................................................................... 142

4.3.6 Effects of BPA on estrogen receptor β expression and activity ............. 144

4.3.7 Effects of DPN and PHTPP on BPA-induced cell proliferation markers. ................................................................................................... 146

4.3.8 Effects of DPN and PHTPP on BPA-induced activation of the shh signaling pathway. .................................................................................. 148

4.4 Discussions ......................................................................................................... 150

4.5 References ........................................................................................................... 153

5 DISCUSSION AND CONCLUSION ........................................................................ 160

5.1 Summary ............................................................................................................. 161

5.1.1 Doses and concentrations of BPA used in in vivo and in vitro experiments ............................................................................................. 161

5.1.2 Prenatal BPA disrupts adrenal steroidogenesis in a sex-specific manner162

5.1.3 BPA stimulates StAR protein expression through estrogen receptor signaling .................................................................................................. 165

5.1.4 BPA stimulates adrenal cortical cell proliferation through ERb-mediated activation of the Shh pathway ................................................. 167

5.2 Future Directions ................................................................................................ 171

ix

5.2.1 To study the effects of prenatal BPA on the other components of the steroidogenic pathway ............................................................................ 171

5.2.2 To determine the precise molecular mechanism behind the effects of BPA on StAR protein levels ................................................................... 172

5.2.3 To determine whether aspects of the signaling pathway identified in vitro can be observed in BPA-exposed mouse adrenal glands ............... 172

5.2.4 To determine the adrenal phenotype in ERa and ERb null mice ........... 173

5.2.5 To determine the effects of BPA analogues on adrenal gland development and function ....................................................................... 174

5.3 Conclusions ......................................................................................................... 176

5.4 References ........................................................................................................... 177

Appendices ...................................................................................................................... 186

Curriculum Vitae ............................................................................................................ 187

x

List of Tables

Table 1.1: Hormone production in the adrenal cortex. ..................................................... 15

Table 1.2: Human and mouse adrenal development. ........................................................ 25

Table 1.3: Effects of BPA on testicular and ovarian StAR expression in vitro. ............... 47

Table 1.4: Effects of BPA on testicular and ovarian StAR expression in vivo. ................ 48

Table 1.5: Effects of BPA on basal corticosterone levels. ................................................ 51

Table 2.1: Primary and secondary antibodies used for western blotting. ......................... 76

Table 3.1: Primary and secondary antibodies used for western blotting. ....................... 100

Table 4.1: Primary and secondary antibodies used for western blotting. ....................... 131

Table 4.2: TaqMan® gene expression assays for the human genes analyzed. ............... 133

Table 5.1: Environmental chemicals that alter adrenal steroidogenesis. ........................ 164

xi

List of Figures

Figure 1.1: Chemical structure of BPA and estradiol.2 ....................................................... 4

Figure 1.2: Prenatal pharmacokinetics of BPA ................................................................. 10

Figure 1.3: Hypothalamic-pituitary-adrenal (HPA) axis. ................................................. 17

Figure 1.4: Adrenal gland development in mice. .............................................................. 23

Figure 1.5: Simplified schematic of the Shh signaling pathway activation. ..................... 29

Figure 1.6: Migration theory of adrenal gland remodeling in mice. ................................. 32

Figure 1.7: Cholesterol transport for adrenal steroidogenesis. ......................................... 36

Figure 1.8: Steroidogenic pathway involved in glucocorticoid synthesis. ....................... 38

Figure 1.9: Synthesis of steroidogenic acute regulatory protein (StAR). ......................... 41

Figure 2.1: Effects of prenatal BPA exposure on adrenal gland weight. .......................... 78

Figure 2.2: Effects of prenatal BPA exposure on plasma corticosterone and ACTH levels.

........................................................................................................................................... 80

Figure 2.3: Effects of prenatal BPA exposure on perilipin protein levels in adrenal glands.

........................................................................................................................................... 82

Figure 2.4: Effects of prenatal BPA exposure on StAR and Cyp11A1 protein levels. .... 84

Figure 2.5: Effects of prenatal BPA exposure on SF-1 protein levels in adrenal glands. 86

Figure 3.1: Concentration-dependent effects of BPA on StAR protein expression. ...... 103

Figure 3.2: Effects of BPA on selected key steroidogenic enzymes. ............................. 105

Figure 3.3: Effects of BPA on ERα and β protein expression. ....................................... 107

Figure 3.4: Effects of ER agonists and antagonist on StAR protein expression. ............ 109

xii

Figure 3.5: Effects of BPA on key StAR transcription factors and StAR mRNA levels.

......................................................................................................................................... 111

Figure 3.6: Effects of BPA on key StAR translation protein and StAR pre-protein. ..... 113

Figure 3.7: Effects of BPA on half-life of StAR protein. ............................................... 115

Figure 4.1: Time- and concentration-dependent effects of BPA on cell proliferation. .. 135

Figure 4.2: Effects of BPA on key cell proliferation factors. ......................................... 137

Figure 4.3: Effects of BPA on selected components of the shh signaling pathway. ...... 139

Figure 4.4: Effects of BPA on activity of the shh signaling pathway ............................. 141

Figure 4.5: Effects of shh inhibition on BPA-induced cell proliferation markers. ......... 143

Figure 4.6: Effects of BPA on estrogen receptor β expression and activity. .................. 145

Figure 4.7: Effects of DPN and PHTPP on BPA-induced cell proliferation markers. ... 147

Figure 4.8 Effects of DPN and PHTPP on BPA-induced shh pathway activation. ........ 149

Figure 5.1: A schematic representation of the postulated molecular pathway by which

BPA stimulates adrenal cortical cell proliferation. ......................................................... 170

Figure 5.2: BPA and its analogues structure 84 ............................................................... 175

xiii

List of Appendices

Appendix 1 ...................................................................................................................... 186

xiv

List of Abbreviations ACA Adrenal adenomas

ACAT Acyl-coenzyme-A-cholesterol-acyl-transferase

ACC Adrenal cortical carcinomas

ACT Adrenal cortical tumors

ACTH Adrenocorticotrophic hormone

AKAP A-kinase anchoring protein

AKT Protein kinase B

AR Androgen receptor

BPA Bisphenol A

BPA-G Bisphenol A glucuronide

BPA-S Bisphenol A sulfate

BPAF Bisphenol AF

BPF Bisphenol F

BPS Bisphenol S

Bw Body weight

C/EBP CCAAT-enhancer binding protein

CBG Corticosteroid binding globulin

CD Cushing’s disease

CDK Cyclin dependent kinase

CHX Cycloheximide

CRH Corticotrophin releasing hormone

CRHR1 Corticotrophin releasing hormone receptor 1

CS Cushing’s syndrome

Cyc Cyclopamine

Cyp Cytochrome P450

DAX-1 dosage-sensitive sex reversal-adrenal hypoplasia

congenital critical region on the X chromosome

factor 1

DHEA Dehydroepiandrosterone

Dhh Desert hedgehog

DOHaD Developmental origins of health and disease

xv

DPN 2,3-bis(4-Hydroxyphenyl)-propionitrile

E Embryonic day

EDC Endocrine disrupting chemical

ELISA enzyme-linked immunosorbent assays

ER Estrogen receptor

ERE Estrogen response element

ERK Extracellular regulated kinase

ERR Estrogen related receptor

EFSA European Food and Safety Authority

FBS Fetal bovine serum

FDA Food and Drug Administration

FGFR Fibroblast growth factor receptor

Foxl2 Forkhead box L2

GPR30 G-protein coupled estrogen receptor

GR Glucocorticoid receptor

GRE Glucocorticoid response element

GS-MS Gas chromatography mass spectrometry

HDL High density lipase

Hh Hedgehog

Hhip Hedgehog interacting protein

HPA axis Hypothalamic-pituitary-adrenal axis

HPLC-MS High performance liquid chromatography–mass

spectrometry

HSD Hydroxysteroid dehydrogenase

HSL Hormone sensitive lipase

IC Inhibitory concentration

ICI ICI 182, 780

IGF Insulin growth factor

Ihh Indian hedgehog

IMM Inner mitochondria membrane

IMS Intra-mitochondrial space

xvi

iNOS Inducible nitric oxidative synthase

LAL Lysosomal acid lipase

LDL Low density lipoprotein

LOAEL Lowest observable adverse effect level

MAPK Mitogen-activated protein kinase

MC2R Melanocortin 2 receptor

MEK Mitochondrial kinases

miRNA micro RNA

MLN64 Metastatic lymph node 64

MRP Multidrug resistance associated protein

NMDRC Non-monotonic dose response curves

NOAEL No observed adverse effect level

OMM Otter mitochondria membrane

PBR Peripheral benzodiazepine receptor

PCNA Proliferating cell nuclear antigen

PHTPP 4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-

a]pyrimidin-3-yl]phenol

PI3K Phosphatidyl-inositol-3-kinase

POMC Proopiomelanocortin

Ptch Patched

PND Postnatal day

qRT-PCR Real time quantitative polymerase chain reaction

SF-1 Steroidogenic factor 1

Shh Sonic hedgehog

SMO Smoothened

SOR StAR overload response

Sp1 Specificity protein 1

SRB1 Scavenger receptor B type 1

SREBP Sterol regulatory element binding protein

StAR Steroidogenic acute regulatory protein

Sufu Suppressor-of-fused protein

SULT Sulfotransferase

xvii

TDI Tolerable daily intake

Tis11b TPA-induced sequence 11b

TSPO mitochondrial transport protein

UGT UDP-glucuronosyltransferases

WNT wingless-related mouse mammary tumor virus

integration site

WT-1 Wilm’s tumor 1

YY1 Ying yang 1

ZF Zona fasciculata

ZG Zona glomerulosa

ZR Zona reticularis

zU Undifferentiated cell zone

1

1 INTRODUCTION

2

1.1 Bisphenol A Bisphenol A (BPA) is a widely used endocrine disrupting chemical (EDC) that has

become a source of major health concerns 1-3. With an estimated production of six billion

tons per year, BPA ranks as one of the most highly produced chemicals 4. Initially, it was

synthesized by Dr. A.P. Dianin in 1891 for use as a synthetic estrogen, however, the

discovery of more potent estrogenic compounds resulted in its discontinuation 2. During

the 1940s and 50s, BPA was identified as a potentially important component of plastics

and began to be utilized in the manufacture of polymers, polyvinyl chloride plastics, and

flame retardant tetrabromobisphenol-A 1-4. Currently, it is commonly found in

polycarbonate plastics, such as plastic containers, baby bottles, plastic water bottles, etc.;

and in epoxy resins, which are used as an internal coating on food and beverage cans 5.

Additionally, BPA is present in medical and dental equipment, thermal paper, and

cardboard and has been detected in soil, water and air samples 2-4,6-8. The BPA used in

packaging contains an ester bond linking BPA monomers onto polymers, making it

susceptible to hydrolysis, thus allowing migration of BPA from the packaging into the

contents 8. BPA has been demonstrated to leach out of plastic containers and liners of

cans into the food or beverage products, a process that is enhanced in acidic or high

temperature environments 2-4,6-8. The most common route of exposure to BPA is through

ingestion, but exposure through dermal routes and inhalation is also possible 5. BPA is

ubiquitous in the environment and is detectable in the urine of 91% of Canadians 9.

Several human epidemiological studies have demonstrated an association between

exposure to BPA and adverse health outcomes that include infertility, reproductive

complications, childhood obesity, childhood asthma, and altered neurological

development in children and adults 10-12. Furthermore, animal studies have shown that

developmental exposure to BPA results in a wide range of adverse health effects

including reproductive, cardiovascular, immunological, metabolic, behavioral, and

neurological disorders, as well as certain cancers in adult offspring, and that many of

these effects are sex specific 2,6,13.

3

1.1.1 Mechanism of Action

BPA is considered an environmental estrogen and an EDC 2-4,6,7 with the ability to bind to

both α and β estrogen receptor subtypes (ER), androgen receptors (AR), G-protein

coupled estrogen receptors (GPER), estrogen related receptors γ (ERRγ) and

glucocorticoid receptors (GR) 5,7. Recent evidence also indicates that BPA exposure

results in adverse effects through pregnane X receptors 14, peroxisome proliferator-

activated receptors 15, thyroid hormone signaling 5,7,15, NF-kB signaling 16, ion signaling 17, and induces pro-inflammatory cytokines and chemokines 18,19. Additionally, prenatal

BPA exposure, even at low doses, has been shown to cause epigenetic alterations,

including DNA methylation and histone modifications 20. BPA’s effects on these

receptors and pathways are based on (1) the presence of the receptors, (2) the level of

expression of these receptors, (3) the dose of BPA, and (4) the level of endogenous

hormones that compete with BPA for binding to these receptors.

1.1.1.1 Estrogen Receptor

Of particular interest are the actions of BPA through the ERs, due to the structural

similarities of BPA to estrogen and its previous use as a synthetic estrogen (Figure 1.1) 15. There are two distinct ER subtypes, ERα and ERβ, which are specific to cell type, with

ERα primarily expressed in reproductive and insulin-sensitive tissues 15. Upon estrogen

binding to the ER, a conformational change occurs and ER translocates to the nucleus of

the cell. In the nucleus, ER can either bind (1) directly to estrogen response elements

(ERE) on promoters of ER target genes to induce gene expression or (2) to transcriptional

coactivators, such as Sp1 and Ap1 to induce gene expression 15,21.

4

Figure 1.1: Chemical structure of BPA and estradiol.2

5

BPA acts as an agonist for ERβ and has dual agonist or antagonist actions for ERα and

has a higher binding affinity to ERβ than to ERα 22-24. Thus, the effects of BPA on ER are

largely dependent on the cell type and ER subtype present 2. However, BPA has been

classified as a weak estrogen based on its low binding affinity for ER compared to

naturally-occurring 17β-estradiol (~10,000-fold lower) 2. This leads to the claim that

BPA will not have a large impact on ER in the presence of endogenous estrogens.

However, additive effects of BPA and estradiol together have been demonstrated 25.

Additionally, the effects of BPA may be explained by the “spare receptor” hypothesis,

which states how a maximal response may be achieved by low concentrations of a

hormone (or EDC), before occupancy of receptors are saturated 22. In addition, BPA has

been shown to have non-genomic actions that are distinct from its actions on classical

ERs 22,26. ER localized to the plasma membrane is known to activate a variety of

pathways depending on receptor and cell type, including the extracellular regulated

kinase/mitogen-activated protein kinase (ERK/MAPK), p38/MAPK, and phosphatidyl-

inositol-3-kinase/protein kinase B (PI3K/AKT) pathways 15. Moreover, BPA has been

demonstrated to activate these non-genomic pathways by acting through membrane ER 15.

1.1.1.2 GPER GPER (also known as GPR30) is a G-protein coupled intracellular membrane receptor

that is activated by estrogen and responsible for rapid estrogen signaling 27. BPA has

been shown to be a strong agonist of the GPER, having non-genomic effects similar to

estrogen 5,28. BPA has a half maximal inhibitory concentration (IC50) of 630 nM for the

GPER, which is 8-50× higher than for classic ERs 28. Additionally, BPA can displace

over 50% of the [3H] E2 binding to the plasma membrane of cells transfected with GPER,

demonstrating BPA’s ability to interfere with estrogen signaling 28.

1.1.1.3 Androgen Receptor

Since BPA also affects the male reproductive system, the results of BPA binding to the

androgen receptor have been investigated. BPA acts as a moderate antagonist of the AR

in numerous cell types 5,7,29,30. BPA has an inhibitory concentration (IC50) value of

3.9×104 nM for the AR in MA-10 cells 29. Additonally, BPA has been shown to

6

competitively bind to the AR and reduce AR nuclear translocation, and therefore its

activity 30.

1.1.1.4 Estrogen Related Receptor γ ERRγ are a subfamily of orphan receptors similar to ERs that can bind to estrogen related

response elements as well as EREs 15. BPA has been shown to be a potent ERRγ agonist 5, with an IC50 value of 13.1 nM, similar to that of the well-known strong ERRγ agonist

4-hydroxytamoxifen 31. Thus, BPA may adversely affect signaling pathways through

ERRγ binding 15. Moreover, there is potential for interaction or interference between

ERRs and ERs during both the developmental period and adulthood 15.

1.1.1.5 Glucocorticoid Receptor

Since BPA has been demonstrated to act as both a GR agonist and antagonist 5,29,32,33, the

actions of BPA on the GR may be tissue and dose specific. The affinity of BPA to the GR

is relatively low, with an IC50 value of 6.7×104 nM in MA-10 cells and has antagonistic

activity toward the GR 29. In the adipose cell line 3T3-L1, BPA displayed agonistic

activity through the GR, which was also demonstrated in an in silico study 32,33.

Additional evidence from our lab has demonstrated that BPA interferes with the

glucocorticoid signaling pathway, resulting in inhibition of glucocorticoid target genes in

A549 lung cells 16.

1.1.1.6 Epigentic Modifications

Recent evidence has emerged, supporting a potential for environmental chemicals to alter

the epigenome during the period of embryogenesis. BPA has been demonstrated to

induce numerous epigenetic alterations in rodent studies, including DNA methylation and

histone modifications 5,34,35. For example, DNA methylation alterations have been

observed to result in a shift in coat color after prenatal BPA exposure in viable yellow

agouti mice 34. Moreover, oocyte maturation in porcine subjects was disrupted by BPA as

a result of DNA methylation and histone modifications 35.

1.1.2 Dose Response and Low Dose Effects Regulatory agencies typically establish the lowest observable adverse effect level

(LOAEL) and/or no observed adverse effect level (NOAEL) for chemicals such as BPA

7

36. This enables the calculation of a reference dose that is considered safe for human

exposure, based on the theory that “the dose makes the poison,” which stipulates that

high doses of a chemical result in a larger physiological effect or in an increase in

potential adverse effects 36. This theory is challenged by the testing of EDCs 36. In

contrast to traditional monotonic response curves, many EDCs including BPA, have non-

monotonic dose response curves (NMDRCs). Traditional monotonic response curves can

be either linear or non-linear, but the slope of the curve always remains in the same

direction 36. NMDRCs are U-shaped or inverted U-shaped, such that the direction or

slope of the curve changes over the range of doses being examined 36. Thus, outcomes of

low-dose exposure to BPA cannot be inferred from results of higher dose exposures 37,

making the traditional development method of LOAEL and NOAEL for BPA

problematic. Since all doses of BPA tested had adverse effects, negating the possibility of

determining a NOAEL value, the reference dose for human exposure of BPA was

calculated using the LOAEL 36. Thus, the widespread use of BPA and consequent

ubiquitous exposure to BPA even at very low doses is of concern.

1.1.3 Pharmacokinetics The metabolism of BPA in humans, rodents, and primates is similar and is thought to

occur through comparable mechanisms 38. Pharmacokinetic studies of BPA indicate that

large amounts of BPA are subject to first pass metabolism in the liver, where it is

conjugated into either the main metabolite, BPA-glucuronide (BPA-G), by uridine 5ʹ-

diphospho-glucuronosyltransferase (UGT) or BPA-sulfate (BPA-S) by sulfotransferase

(SULT). Among UGTs, UGT2B15 shows the highest activity for conjugating BPA into

BPA-G in human liver microsomes 39. In both rodents and humans, glucuronidation and

sulfation produce inactive hydrophilic BPA metabolites that is excreted in the feces 38,40.

However, unconjugated BPA is cleared by the kidneys and eliminated in the urine of

primates, but is excreted primarily in the feces of rodents 38. This difference in route of

elimination raises the question of potential differences in metabolism and circulating

levels of BPA. Taylor, et al. 38 demonstrated that oral administration of BPA results in

similar levels of unconjugated BPA in the circulation and identical rates of clearance in

non-human primates and rodents. However, in pregnant rhesus monkeys, the systemic

bioavailability or the percent of unconjugated BPA that reaches the systemic circulation

8

of BPA is considerably lower; only 0.48% 41. Rapid conjugation of BPA was

demonstrated when pregnant rhesus monkeys were injected with 100 µg/kg bodyweight

(bw) of unconjugated BPA, and at the five-minute initial sampling point, 87% of the BPA

detected was in the conjugated form. The presence of unconjugated BPA in the blood

demonstrates either (1) incomplete first pass metabolism, (2) a bypass of first pass

metabolism or (3) the potential for deconjugation of BPA metabolites into unconjugated

BPA 4. Indeed, evidence suggests that in the intestine and colon, conjugated BPA can be

deconjugated back into active BPA in rodents 4. Clearly, additional investigation is

required to determine the pharmacokinetics of BPA and the potential differences in

experimental animal models versus human exposure to comprehend the risks of BPA

exposure.

During pregnancy, the placenta plays a critical role in controlling potential toxins that can

reach the fetal circulation (Figure 1.2). Detectable levels of BPA were present in the

placentae of rhesus monkeys given a single injection of 100 µg/kg bw BPA 41 and of that,

29% was unconjugated BPA 41. Using a placental transfer model, 27% of unconjugated

BPA was shown to cross from the maternal circulation of the placenta to the fetal

circulation, indicating that unconjugated BPA can freely cross the placenta 42. Moreover,

this model demonstrated that the placenta plays no role in conjugating active BPA into

BPA-G, since BPA-G was detected in neither the maternal nor the fetal placental

circulation 42. In addition to passive diffusion of BPA, there are active placenta transport

proteins such as organic anion transporting polypeptide (Oatp) and multidrug resistance

associated protein (Mrp) transporter family members that aid in the transport of

conjugated BPA 40,43,44. BPA-G has been shown to cross into the placenta via the

Oatp4a1 transport and then into fetal circulation by Mrp1 transporter 40,44. Thus, BPA has

the potential to reach fetal circulation in both conjugated and active forms.

The fetal liver is reported to have a 36-fold lower level of UGT2B15 than the adult liver,

suggesting that the fetus has a decreased potential to metabolize BPA 40,45,46. While BPA-

G is considered to be inactive, β-glucuronidase in the fetal liver can deconjugate BPA

into the active form 40,47. Thus, the risk of BPA to the fetus is compounded by its ability

to de-conjugate BPA-G, as well as by its undeveloped drug detoxifying system 40,46.

Additionally, since BPA-G is water soluble and cannot cross back through the placenta

9

into maternal circulation after excretion in the fetal urine, it may become trapped in the

amniotic fluid where it has the potential to continually re-enter fetal circulation 47. Thus,

there is growing concern about the risk of the developing fetus to prenatal exposure to

BPA.

10

Figure 1.2: Prenatal pharmacokinetics of BPA

Absorption of BPA occurs in the maternal intestines, BPA can either (1) be metabolized

by the maternal liver to BPA-G, by UGTs, and then transported across the placenta by

transport proteins or (2) freely diffuse across the placenta unconjugated and reach the

fetal circulation. Conjugated BPA in the fetus can be unconjugated by b-Gase by the fetal

liver to unconjugated BPA. Unconjugated BPA can then reach target fetal organs and

accumulate in the fetus. Abbreviations: BPA, Bisphenol A; UGT, uridine 5’-diphospho-

glucuronosyltransferase; BPA-G, BPA-glucuronide; b-Gase, β-glucuronidase.

11

1.1.4 Human Exposure to BPA It is estimated that 90-95% of people have detectable levels of BPA in their urine,

demonstrating the universality of BPA exposure 9,15. Ninety percent of BPA exposure is

thought to result through food and drink exposure, with the remaining exposure through

dust, dental products and surgery, and dermal contact 15. Health Canada has designated

the level of tolerable daily intake (TDI) of BPA to be 25 µg/kg bw per day for the

average Canadian adult, and in 2010 banned the use of BPA in baby bottles and food

containers 48,49. Even with such regulations in place, estimated BPA daily intake levels in

Canadian infants are reported as 0.22-0.33 µg/kg bw/day and levels in adults have been

estimated at 0.052-0.081 µg/kg bw/day 50. In the United States, the Food and Drug

Administration (FDA), eliminated the use of BPA in epoxy resins of baby food

containers due to marketplace demands in 2013 49. However, the FDA continues to state

that BPA is safe for consumers at their current levels 49. Similarly, the European Food

and Safety Authority (EFSA) reported there was no health concern of dietary BPA for

any age group 49, but as a precautionary measure, reduced the levels of safe BPA

exposure from 50 µg/kg bw/day to 4 µg/kg bw/day, and banned the sale of baby bottles

containing BPA 49. Nonetheless, government agency regulations aimed at preventing

exposure to BPA in infants and young children do not address the issue of fetal exposure

to BPA during pregnancy via maternal sources, which has been shown to have

permanent, long lasting effects on human health 11,12.

1.1.4.1 Developmental Origins of Health and Disease Over thirty years ago, David Barker first considered the possibility that poor maternal

malnutrition resulting in low birth weight of the fetus led to premature death due to

metabolic and cardiovascular complications later in life for the offspring 51. This concept

was expanded to include early life exposure to environmental toxins that can lead to

subtle changes during development that lead to dysfunction and/or diseases later in life

and is now referred to as the Developmental Origins of Health and Disease (DOHaD)

hypothesis 51. Application of this hypothesis to BPA implies that prenatal exposure to

BPA can have long lasting effects that span a lifetime due to alterations in gene and

protein expression that occur during the critical period of organ development 51,52.

Epigenetic alterations in the fetus are also thought to play an important role in the

12

susceptibility to dysfunction and/or disease later in life 52. Furthermore, many of these

developmental effects can be sex-specific and are often irreversible 51. As such,

developmental exposure to environmental stressors (altered nutritional status,

environmental chemical, stress, etc.) can have lasting effects on the offspring, which is

not yet fully understood.

1.1.4.2 BPA during Pregnancy Dynamic changes occur in drug metabolism and transport during pregnancy, and thus

BPA metabolism may vary in pregnant versus non-pregnant populations 40,53. During

pregnancy, women have increased plasma volume and clearance as well as altered

metabolism depending on the specific enzymes involved 40,53. Consequently, there is a

possibility of an increased half-life of BPA in the maternal circulation thereby increasing

potential fetal exposure 47.

While the placenta is considered a protective barrier between the mother and fetus, many

environmental chemicals pass through the placenta due to their high lipophilic properties 54. Furthermore, the presence of both ERα and ERβ on the placenta make it vulnerable to

estrogenic environmental contaminants such as BPA 54. The ban of BPA from baby

products (e.g. baby bottles and toys) does not reduce the risk of BPA exposure to the

developing fetus 11,12. Indeed, pregnant German women were found to have plasma levels

of BPA as high as 9.2 ng/mL, and their fetuses had plasma levels of BPA averaging 12.7

ng/mL, proving that BPA crosses the placenta 55. This finding was supported by North

American and Canadian studies that determined BPA levels in maternal serum to be

between 0.5-22.3 ng/mL 56, and BPA levels in urine from pregnant women to be between

0.16-43.20 ng/mL, respectively 57. BPA has also been detected in cord blood, amniotic

fluid, and breast milk 55,58-60. Taken together, these studies show cause for concern about

in utero exposure to BPA during fetal development 13.

1.1.4.3 Fetal Exposure to BPA One of the major functions of the placenta is to act as a barrier preventing xenobiotics in

the maternal circulation from reaching the fetus 42. However, the placenta appears to be

ineffective at preventing the transfer of BPA, since unconjugated BPA readily crosses the

placenta by passive diffusion in both directions 42,61. While members of the BPA

13

metabolizing enzyme UGT and SULT families are expressed in the placenta, they have

negligible efficacy in conjugating BPA in this milieu 42. In addition to its detection in

cord blood, fetal blood, placental tissues, and amniotic fluid 3,42,55, BPA was found in

fetal tissues, including fetal liver samples (9.02 ng/g unconjugated BPA, 25.8 ng/g total

BPA) collected from the greater Montreal area between 1998 and 2008 62. Thus, BPA is

readily crossing the placental barrier and reaching the fetus.

Various animal models employed to examine the effects of fetal exposure to BPA have

demonstrated that fetal BPA exposure has a wide range of adverse effects including

reproductive, cardiovascular, immunological, metabolic, behavioral, and neurological

disorders; contributes to the development of certain cancers in adult offspring; and that

many of these effects are sex-specific 2,6,13. In mouse models, pre- and post-natal

exposure to low doses of BPA affected the organization of the central nervous system and

neurotransmitter receptor systems, resulting in reduced and/or reversed sexual differences

in the emotional behavior of offspring 63,64. Moreover, in this model, low doses, but not

high doses of BPA, resulted in metabolic disruption (increased body weight, adipocyte

number, abdominal fat, insulin levels, and impaired glucose tolerance) in male offspring 63,65. Our laboratory has demonstrated that prenatal exposure to BPA impairs the

development of the fetal liver 66, pancreas 67 and lungs 68. Therefore, due to the

ubiquitous nature of BPA, there is increasing concern for the potential long-term

consequences of developmental exposure to BPA.

1.2 The Adrenal Gland The adrenal glands are an important endocrine organ that synthesizes hormones in its

cortex and medulla 69,70. The adrenal cortex produces steroid hormones during fetal and

adult life, including glucocorticoids, aldosterone, progesterone, and precursors of

testosterone and estradiol 69,70. These hormones are produced via the steroidogenic

pathway, which starts with cholesterol and involves a number of cytochrome P450

enzymes and hydroxysteroid dehydrogenases 69,70.

The adult adrenal cortex is divided into three zones: the zona glomerulosa (ZG), zona

fasciculata (ZF) and the zona reticularis (ZR) 70 (Table 1.1). The outermost adrenal zone,

the ZG, secretes aldosterone and is a key component of the renin-angiotensin-aldosterone

14

axis, which is responsible for the regulation of water balance 71,72. Aldosterone

transcriptionally regulates a number of proteins and enzymes involved in maintaining

water and sodium balance, and potassium excretion in the kidney 71,72. Cells of the ZG

contain numerous mitochondria, and some cytoplasmic lipid droplets 71. The middle

adrenal cortex zone, the ZF, secretes glucocorticoids, a key hormone in the

hypothalamic-pituitary-adrenal (HPA) axis 71. Cells in the ZF are arranged in bundles,

called fascicles that are surrounded by numerous capillaries 71. These cells contain large

numbers of mitochondria, as well as prominent smooth endoplasmic reticulum and large

lipid droplets used for steroidogenesis 71. The ZR, the innermost adrenal cortex zone,

secretes dehydroepiandrosterone (DHEA), a precursor of testosterone and estrogens 71.

Cells of the ZR are similar in shape and size to the ZF but have more lysosomes and

fewer lipid droplets 71. The adrenal medulla is composed of chromaffin cells that

synthesize epinephrine and norepinephrine 73. All adrenal gland hormones play various

critical roles in maintaining homeostasis. However, this thesis specifically focuses on the

regulation of adrenal glucocorticoid synthesis.

15

Table 1.1: Hormone production in the adrenal cortex.

Zona Glomerulosa Zona Fasciculata Zona Reticularis

Stimulus Angiotensin II ACTH ACTH

Primary Receptor

Angiotensin II receptor MC2R MC2R

Hormone Product

Mineralocorticoids (Aldosterone)

Glucocorticoid (cortisol/corticosterone)

Androgens (DHEA)

Function Regulation of intravascular volume

Glucose homeostasis Stress response

Immune response Adrenarche

Abbreviations: ACTH, Adrenocorticotropic hormone; MC2R, melanocortin 2 receptor; DHEA,

dehydroepiandrosterone.

16

1.2.1 The HPA Axis The production of glucocorticoids is regulated by the HPA axis (Figure 1.3) 74,75. Upon

stimulation, corticotrophin releasing hormone (CRH) is synthesized and secreted from

the paraventricular nucleus in the hypothalamus into the hypophyseal portal vessels to be

transported to the anterior pituitary gland 75. Binding of CRH to the CRH receptor 1

(CRHR1) in the anterior pituitary gland induces synthesis of adrenocorticotrophic

hormone (ACTH) from the prohormone proopiomelanocortin (POMC) 75. ACTH is

released into the systemic circulation where it binds melanocortin 2 receptor (MC2R), a

G-protein coupled receptor expressed on the adrenal cortex, to stimulate steroidogenesis 76. The stimulatory effect of ACTH can increase steroidogenesis in a number of ways

including (1) promoting adrenal cortex growth over the long term 77; (2) promoting the

up- regulation of its receptor MC2R 78; (3) increasing the presence of the scavenger

receptor class B member 1 (SRB1) and low-density lipoprotein (LDL) receptors, thereby

enabling enhanced uptake of cholesterol 78; and (4) up-regulating key steroidogenic

enzymes such as cyp11A1 77,78 and steroidogenic acute regulatory protein (StAR) 78.

The HPA axis is tightly regulated by a glucocorticoid negative feedback mechanism 75.

Glucocorticoids produced from the adrenal gland provide a negative feedback signal to

the hypothalamus and pituitary gland to inhibit further glucocorticoid production through

altering transcription of HPA components upon binding to glucocorticoid responsive

elements (GREs) or interaction with various transcription factors 75. Disruptions in the

development and formation of the HPA axis pathways during the critical window of fetal

development has long lasting health consequences that extend into adulthood, including

metabolic syndrome 79 and anxiety/mood disorders 80,81.

17

Figure 1.3: Hypothalamic-pituitary-adrenal (HPA) axis.

Hypothalamus releases CRH binding to CRHR-1 receptors on the anterior pituitary gland.

Binding of CRH stimulates the release of ACTH which binds to MC2Rs on the adrenal cortex,

causing the release of glucocorticoids; cortisol in humans and corticosterone in mice.

Glucocorticoids will negatively feedback to both the hypothalamus and anterior pituitary gland to

regulate its expression. Abbreviations: CRH, corticotrophin releasing hormone; CRHR-1,

corticotrophin releasing hormone receptor-1; ACTH, adrenocorticotrophic hormone; MC2R,

melanocortin 2 receptor.

18

1.2.2 Physiological Function Glucocorticoids are essential in maintaining whole body homeostasis through their

various actions in numerous tissues. Glucocorticoids play a major role in glucose

metabolism in stressful environments through increasing serum glucose and amino acids

by (1) increasing catabolism of muscle to increase circulating amino acids; (2) increasing

amino acid uptake in the liver to increase gluconeogenesis and glycogenesis; and (3)

decreasing peripheral glucose uptake in muscle and adipose tissue 78,79,82,83. Additionally,

glucocorticoids increase general catabolism by increasing lipid hydrolysis and increasing

fatty acids and by increasing bone and connective tissue catabolism, which may result in

osteopenia, and thinning of skin and support structures 78,79,82-84. Glucocorticoids also

play an important role in the immune system by suppressing the inflammatory response

while promoting anti-inflammatory actions 78,85.

1.2.2.1 Cushing’s Disease Cushing’s disease (CD) is an endocrine disorder characterized by the overproduction of

ACTH due to a pituitary tumor, commonly an adenoma, which results in overstimulation

of the adrenal gland 86-89. While CD is the most common form of Cushing’s syndrome,

other causes include excessive use of glucocorticoids 86-89. Cushing’s syndrome is defined

by increased cortisol in both the serum and urine, with a disruption of the HPA axis and

cortisol circadian rhythm 86. The prevalence of CD is reported as 40 cases per million,

and has highest prevalence in women aged 40-60 years old 86. Symptoms of excessive

glucocorticoids include increased weight gain, fatigue, insulin resistance, skin thinning,

and bruising 86,87,89. The wide range of comorbidities associated with CD includes

hypertension, diabetes mellitus, dyslipidemia, osteoporosis, depression, impaired sexual

function in men, menstrual disorders in women, and infertility in both men and women 86,88. Additionally, patients with persistent or recurring CD or excessive glucocorticoid

production have increased risk of mortality 86. Untreated CD has a five-year survival rate

of less than 50% 89; with the most common causes of mortality being cardiovascular

disease and infection 86,87. Patients with CD report a significant decrease in their quality

of life, physically, mentally, and emotionally 86-88. Treatment for CD varies, depending

on the source of the condition, but the first line of treatment is surgery (pituitary and/or

adrenal gland) 86,87. Pharmacological therapies, including steroidogenesis inhibitors (e.g.

19

ketoconazole, metyrapone, etomidate) and glucocorticoid receptor antagonists (e.g.

mifepristone) are used preoperatively or for reoccurrences 87. Treatment of CD may

reduce symptoms of the disease, but comorbidities may be irreversible, therefore

potential risk persists throughout life 88.

1.2.2.2 Addison’s disease Opposite to adrenal over-activity is adrenal insufficiency, often known as Addison’s

disease, which is a rare chronic endocrine disease that results in loss of adrenal function,

with a subsequent decrease in adrenal production of glucocorticoids, as well as

mineralocorticoids in certain cases 90-92. Adrenal insufficiency is classified as primary or

secondary adrenal insufficiency 90,91. Primary insufficiency, which affects 0.01% of the

population, results from direct inhibition of glucocorticoid production from the adrenal

gland. Primary adrenal insufficiency can be caused by autoimmune adrenalitis, infectious

adrenalitis (e.g. AIDS, tuberculous adrenalitis), bilateral adrenal hemorrhage, adrenal

infiltration, adrenalectomy, drug-induced adrenal insufficiency, or various genetic

disorders (mutations in any of the adrenal steroidogenic enzymes, regulatory transcription

factors, or receptors) 90,91. Secondary adrenal insufficiency has a prevalence of 1 in 5,000

and is a result of decreased stimulation of adrenal gland steroidogenesis due to decreased

ACTH levels 90,91. This can be a result of tumors of the pituitary gland or hypothalamus,

chronic glucocorticoid use, head trauma, isolated congenital ACTH deficiency,

proopiomelanocortin-deficiency, or combined pituitary-hormone deficiency 90,91. Adrenal

insufficiency presents with a range of symptoms depending on whether the adrenal

insufficiency involves decreased glucocorticoids, mineralocorticoids, and/or androgens 92.

Common clinical manifestations of both primary and secondary adrenal insufficiency

include fatigue, anorexia, muscle weakness, weight loss, light-headedness, nausea,

vomiting, headache, sweating, salt craving, and, in women, dry itchy skin and loss of

libido 90-92. In addition, adrenal insufficiency can result in an adrenal crisis 90,92, which is a

severe lack of glucocorticoids during extreme stress, infection, or trauma, and can be life

threatening 90. Monitoring of glucocorticoid levels and adjustment during times of stress

can prevent adrenal crisis, but mortality rates are still 1.5-2 fold higher in patients

suffering from adrenal insufficiency 90. Current treatment of adrenal insufficiency is to

compensate for the glucocorticoid and mineralocorticoids deficiency (only in primary

20

adrenal insufficiency), with multiple daily tablets of hydrocortisone or prednisone and

fludrocortisone in doses that mimic normal hormone secretion patterns 91,92. Primary

adrenal insufficiency results in a decrease in lifespan of 3.2 years in women and 11.2

years in men, compared to the general population, mainly due to increased acute adrenal

failure, infections, and sudden death 92. People suffering from adrenal insufficiency report

a decreased perception of health status and quality of life 92.

1.2.2.3 Adrenocortical Cancer Adrenal cortical tumors (ACTs) are classified as either malignant adrenal cortical

carcinomas (ACCs) or benign adrenal adenomas (ACA) 93,94. Small ACA’s are relatively

common, affecting about 3-10% of the population 94. In contrast, ACCs are a rare form of

cancer with an annual incidence rate of between 1 to 2 million 93,94. These tumors present

with an aggressive phenotype and the patients have a poor prognosis 94,95. They are

characterized by altered production of steroid hormones, uncontrolled tumor growth and

metastases to other tissues 95. Between 50-80% of patients present with hypercortisolism

and 40-60% of patients present with excess adrenal androgen production 94.

Numerous genetic mutations and alterations have been linked to the development of

ACTs 95. Mutations in TP53 predisposes children to pediatric ACTs (as well as other

conditions). This is particularly relevant in southern Brazil, which has a 10-fold higher

rate of pediatric ACTs due to mutations in TP53 96. Levels of insulin growth factor II

(IGFII) is commonly used as an ACC marker due to its overexpression in 90% of ACCs 95,97. Alone, mutations that result in increased IGFII levels are not a significant factor for

ACC development, but these mutations may contribute to ACC progression in

combination with other factors 95,98. Activating mutations of b-catenin, leading to

increased activation of Wnt signaling, has been detected in ACC patients 95,99.

Additionally, mutation in genes shown to regulate or be involved in Wnt signaling

potentially lead to an increase in ACTs 95. Mutations in other genes including multiple

endocrine neoplasia 1 (MEN1), mutL homolog 1 (MLH1), mutS homolog 2 (MSH2),

mutS homolog 6 (MSH6), post meiotic segregation increased 2 (PMS2) and post meiotic

segregation increased 2 (PRKAR1A) have all been observed in patients with ACCs 95.

21

Alterations in gene expression in ACTs are commonly divided between ACC and ACA 95. Changes in gene expression frequently seen in ACC are the overexpression of cell

proliferation and cell cycle genes, such as cyclin E1, cyclin E2, and cyclin dependent

kinase 2 and 4 (CDK2 and 4) 95,100. Additionally, ACCs have alterations of steroidogenic

enzymes, including cyp11A1, StAR, cyp17A1, while these enzymes are generally

upregulated in ACAs 95. Other pathways known to be affected in ACTs include, IGFII 101, sonic hedgehog (Shh) 102,103, Wnt 95,99, fibroblast growth factor receptor (FGFR) 101,

and retinoic acid signaling pathways 101.

1.2.3 Adrenal Development The adrenal gland is developed from two different cells types: the adrenal medulla arises

from neural crest cells, while the adrenal cortex arises from coelomic epithelium

(urogenital ridge) (Figure 1.4) 104. The adrenogonadal primordium develops from the

coelomic epithelium, with the presence of developmental regulatory factors, Wilm’s

tumor 1 (WT-1), and wingless-related mouse mammary tumor virus integration site 4

(WNT4) 104. In mice, at embryonic day 9 (E9), the key developmental factors

steroidogenic factor-1 (SF-1) and dosage-sensitive sex reversal-adrenal hypoplasia

congenital critical region on the X chromosome factor 1 (DAX-1) can be detected in the

adrenogonadal primordium 104,105. Migration of adrenal progenitor cells from the

adrenocortical primordium, happens in parallel to an upregulation of SF-1 71,95. The

importance of SF-1 in adrenal development is demonstrated in studies utilizing knockout

mice, where SF-1-/- mice lacked adrenal glands and died at birth due to adrenal

insufficiency 106. The expression of steroidogenic enzymes begins at E11 in mice,

indicating the possibility of steroidogenesis 105. At approximately E12-14, neural crest

cells migrate and disperse into the developing adrenal gland, preceding their development

into the chromaffin cells of the adrenal medulla 107. Encapsulation of the adrenal cortex is

completed by E14.5 104,105, and adrenal cortex zonation is completed between postnatal

days (PND) 1-7 in mice 105. The formation of the X-zone surrounding the adrenal

medulla develops between PND10-14 in mice, and continues to proliferate until PND21 105. The function of the X-zone and its presence postnatally is still not fully understood 105. In male mice, the X-zone will disappear at sexual maturity, whereas in females, it

22

remains until the first pregnancy 105. Encapsulation of the medulla by a fibrous tissue

layer is completed only after complete regression of the X-zone 105.

23

Figure 1.4: Adrenal gland development in mice.

Urogenital ridge separates into either the (2a) fetal kidney or (2b) the adrenogonadal

primordia, which derives into the (3a) bipotenial gonads or the (3b) adrenal primordia,

where steroidogenic enzymes are first detected at E11. Followed by (4) neural crest cells

migration at E12-14. (5) Zonation occurs at PND1-7, and (6) X-zone development at

PND14-21. (7) Medulla encapsulation and X-zone regression occurs at sexual maturity in

males and at the first pregnancy in females. Abbreviation: E, embryonic day; PND,

postnatal day.

24

While development of the adrenal gland in humans differs from that of the mouse in

terms of timing of developmental processes, the factors responsible for adrenal gland

development are thought to be similar between the two species (Table 1.2) 105. In

humans, migration of coelomic epithelial cells starts during week 5-7 of pregnancy, and

the formation of the fetal zone, which produces DHEA, begins at week 7 105,107. Despite

considerable differences in timing of development, the fetal zone in humans is thought to

be equivalent to the X-zone in mice 105. The adrenal primordia develops at around 8

weeks 105,107. At 9 weeks, migration of mesenchymal capsular cells to encapsulate the

adrenal cortex and neural crest cells to form the adrenal medulla 95,105. Regression of the

fetal zone takes place shortly after birth (postnatal weeks 1-6), followed by the

encapsulation of the medulla (postnatal months 12-18) 105,107. Complete adrenal cortex

zonation follows later, around the time of puberty in both males and females (10-20

years) 105.

Estrogen and ERs are thought to play critical roles in the development of the adrenal

gland 108. The discovery of both ERa and ERb in the fetal adrenal cortex suggests a role

for estrogens in regulating adrenal development and function 108. Upon binding to Ers,

estrogens induce direct and indirect effects in the fetal adrenal gland, affecting sensitivity

and responsiveness to ACTH, and altering the synthesis of DHEA 108,109. However, the

role of estrogens or Ers in programming glucocorticoid synthesis in the fetal adrenal

gland remains elusive.

25

Table 1.2: Human and mouse adrenal development.

Human Mouse

Migration of coelomic epithelial cells 5-7 weeks E9

Development of the adrenal primordia 8 weeks E11

Migration of neural crest cells 8 weeks E12-14

Regression of fetal zone/X-zone Postnatal week 1-6 PND35 (males), after

first pregnancy (females)

Medulla Encapsulation Postnatal month 12-18 PND35

Adrenal Cortex Zonation 10-20 years PND1-7

Abbreviations: E, embryonic day; PND, postnatal day.

26

1.2.4 Adrenal Remodeling and Growth Differentiation and renewal of the adrenal cortex zones is not fully understood. However,

a few theories have been proposed for adrenal zonation: (1) Migration Theory that

hypothesizes the centripetal proliferation of cells from the outermost zone, ZG, towards

the ZF, then the ZR and finally undergoing apoptosis at the edge of the adrenal medulla;

(2) Transformation field theory that postulates the presence of two transformation fields

between the ZG and ZF and between the ZF and ZR, where proliferation and

differentiation occurs; (3) Zonal theory that proposes that all proliferation in each zone

comes from cells located in the same zone 110,111. The most probable theory is migration

theory 95,107,111,112, which posits that proliferation begins with specialized cells located

peripherally in the cortex and that these cells will transit inwards through the cortex

layers, and finally to the medulla border where they undergo apoptosis, a process referred

to as centripetal displacement 111,112. This process and the location of specific stem cells

varies in rats, where these cells are likely located in an undifferentiated cell zone (zU),

between the ZG and ZF 111. Continued remodeling and growth is essential in the adrenal

gland after birth and throughout life 112-114. These progenitor cells are not only important

for the development of the adrenal cortex, but also are involved in adrenal cortex

remodeling in adults 112-114.

27

1.2.4.1 Hedgehog Signaling The Hedgehog (Hh) signaling pathway is essential in embryogenesis, adult remodeling

homeostasis, and carcinogenesis in a variety of tissues 115. The Hh signaling pathway

regulates genes involved in proliferation, the stem-cell signaling network, stem-cell

markers, survival, and epithelial-to-mesenchymal transition 115.

The Hh secretory proteins were first discovered in Drosophila for their role in specific

embryonic segmentation 116. The three main mammalian HH genes are Sonic hedgehog

(Shh), Desert hedgehog (Dhh), and Indian hedgehog (Ihh). All are critical for

development, as demonstrated in loss of function studies that result in structural

abnormalities and malformations 116,117. Dhh is localized mainly to the gonads, including

Sertoli cells and granulosa cells. While Dhh-/- mice are viable and do not have a notable

phenotype, males are infertile 117. Ihh expression is limited to the primitive endoderm and

prehypertrophic chondrocytes, resulting in 50% lethality in knockout mice, with

surviving Ihh-/- mice having bone abnormalities, including cortical bone defects and

aberrant chondrocyte development 117. Shh is more broadly expressed both during

embryogenesis and later life 117. Mutations in Shh have been shown to cause cyclopia, as

well as defects in the foregut and ventral neural tube patterning 117. Additionally, defects

present later in life as malformations of the limbs, ribs, and lungs 117.

All Hh secretory proteins bind to the Hh receptors, Patched (Ptch) 1 and 2 to activate the

signaling pathway (Figure 1.5). Patched is a 12-pass transmembrane receptor located on

the primary cilium of target cells. Co-receptors of the Hh signaling pathway include

CDON, BOC, and GAS1 115. When not bound, Ptch inhibits another transmembrane

protein, smoothened (SMO), by keeping it sequestered in the plasma membrane 117-119.

SMO is a 7-pass G-protein coupled receptor that is also located in the plasma membrane

of the primary cilium. In the plasma membrane SMO is tethered to a complex containing

the key Shh transcription factors Gli 117-119. When the Shh pathway is activated, Hh

prevents Ptch from inhibiting SMO and enables the translocation of SMO into the

cytoplasm 117-119. Upon SMO translocation, the complex containing the Gli transcription

factors are released. In mammals, there are three Gli transcription factors (Gli1-3), with

various activities 117-119. Gli1 and Gli2 are primarily activators of the Shh signaling

pathway and have similar roles 117-119. However, Gli1 is also responsible for positive

28

feedback of the Shh signaling pathway, and is a direct transcriptional target of Shh

activation, to extend cellular response 117-119. The activation of SMO blocks proteolysis of

Gli1 and 2, which leads to accumulation of the full-length activator forms of Gli1 and

Gli2, ultimately leading to transcription of target genes 117-119. Gli3 is primarily a

repressor; in the absence of Shh, Gli3 is cleaved into an active repressor form, to inhibit

transcription of target genes, but the presence of Shh prevents Gli3 cleavage and thus

inhibits Gli3 activity 117-119. Additional factors known to be involved in the regulation of

the Shh pathway include suppressor-of-fused protein (Su(fu)) and hedgehog interacting

protein (Hhip), both of which attenuate Shh signaling 115,118.

29

Figure 1.5: Simplified schematic of the Shh signaling pathway activation.

The secretory protein Shh, acts in an autocrine/paracrine fashion and binds to Ptch1

receptor, preventing Ptch1 from inhibiting SMO. SMO is then released from the plasma

membrane into the cytoplasm, leading to the release of key Shh transcription activators

Gli1 and Gli2. Gli1/2 translocate to the nucleus where they bind to the promoters of

target genes to regulate gene expression. Abbreviations: Shh, sonic hedgehog; Ptch1,

Patched 1; SMO, smoothened.

30

Due to the importance of Shh signaling in development and organ remodeling later in

life, the Shh signaling pathway has been investigated in both rodent and human adrenal

glands. Expression of mRNA for Shh, Ptch1, and Gli1 begins at E12.5 in the mouse

adrenal cortex, and is localized in clusters at the periphery of the adrenal cortex, which

continues throughout development 112,114,120. Shh, Gli1, 2, and 3 proteins are detected

throughout development and postnatally in the fetal human adrenal gland, with higher

expression than is found in adult human adrenal glands 102. In contrast, Werminghaus et

al. demonstrated undetectable levels of Shh protein in both normal adult adrenal glands

and adrenocortical carcinomas and adenomas 103. However, protein levels of Gli1 were

detectable in all adrenal cortex zones, but concentrated in the subcapsular area of the ZG

of human adult adrenal glands, and was not detectable in adrenocortical carcinomas or

adenomas 103. While additional studies have also detected mRNA for Shh, Gli1, 2 and 3,

Ptch1 and SMO in human adrenal glands, adrenocortical carcinomas and adenomas 102,103. Additionally, there was an upregulation of SHH in both cortisol producing and

non-cortisol producing adrenal adenomas compared to normal adrenal tissues, suggesting

that Shh activation is involved in adrenal tumorigenesis 102,103. Moreover, Shh, Gli1,

Ptch1 and SMO mRNA has been found in human adrenal cortical carcinoma cell lines,

H295R and H295A102,103. The presence of Dhh or Ihh in the adrenal glands has yet to be

determined 113.

Shh and Shh pathway components (Ptch1, SMO, Gli1) mRNA and protein are localized

in the outer cortex cells, which do not express the steroidogenic enzymes cyp11b1 or

cyp11b2, during early organogenesis and throughout adulthood in mice 112. This indicates

the presence of a specialized population of cells in the adrenal cortex that lacks the ability

to produce steroid hormones 112. This demonstrates the essential role of Shh signaling in

the development and expansion of the adrenal cortex 114, and supports the theory of

adrenal growth and remodeling through a centripetal displacement process, where Shh

containing cells are progenitor cells that differentiate into steroidogenic cells (Figure 1.6) 112,113. Indeed, previous studies using genetic lineage analyses performed using a

constitutive Cre model, demonstrated that Shh-positive cells give rise to cortex cells in all

zones except the medulla 112. Moreover, lineage analysis in adults, shows that cells

31

transition from the outer ZG to the ZF, demonstrating that Shh marks progenitor cells in

the adrenal cortex during development and remodeling in adults 112,113.

32

Figure 1.6: Migration theory of adrenal gland remodeling in mice.

Progenitor cells (purple), which are Shh+/Sf-1+/cyp11B2-, signal to Gli1+/cyp11B2-

capsule cells (red), to differentiate into functional ZG steroidogenic cells (orange). The

migration theory suggests a centripetal displacement process where ZG cells move

inward and differentiate into ZF cells (blue) and on to X-zone cells (dark red), before

undergoing apoptosis at the medulla border. Abbreviations: Shh, sonic hedgehog; Sf-1,

steroidogenic factor-1; ZG, zona glomerulosa, ZF, zona fasciculata.

X-zone

Progenitor cells

ZF

Medulla

ZG

Capsule

33

Gli3 mutation was found to be lethal in embryonic mice and to have an adrenal aplasia

phenotype 121. However, this phenotype was not observed by Laufer, et al. 113. Adrenal

Shh conditional knockout mice, created with a Sf-1-cre driver, exhibit severe hypoplasia

and underdevelopment of the adrenal gland, but have no changes in zonation or

differentiation of the adrenal cortex 112,114,120. There was a significant decrease in both the

thickness of the adrenal cortex and the capsule in these mice 112,114. These effects were

visible as early as E13.5, with no visual changes to the adrenal medulla 112,114,120.

However, despite the reduction in cortex size, the expression of steroidogenic enzymes

was unaltered in Shh-/- mice 112,114,120. Corticosterone levels were normal until

approximately one year of age, when they became reduced along with an increase in

ACTH plasma levels 114. Moreover, Shh-/- mice had reduced levels of proliferating cells in

their adrenal cortex, with no change in apoptosis levels 114. In H295R cells, blocking Shh

signaling with the antagonist cyclopamine resulted in decreased proliferation, and

decreased production of aldosterone and DHEA 103.

1.2.4.2 Wnt-1 Signaling In both fetal and adult adrenal glands, the Wnt/b-catenin signaling pathway is critical for

adrenocortical homeostasis 122. b-catenin is present in the fetal adrenal cortex, and is

localized to the ZG subcapsule 122. Mice null for b-catenin in SF-1 expressing adrenal

cortex cells, show abnormal adrenal development starting at E12.5, resulting in adrenal

failure 123. b-catenin Sf-1-cre mice, which expressed b-catenin in approximately half of

their adrenal cortex cells, developed normally 123. However, a thinning of the adrenal

cortex and decreased steroidogenic function was observed starting at 30 weeks of age 123.

Additional evidence for the role of Wnt-1/b-catenin signaling in adrenal development and

function is demonstrated when the signaling pathway is over-activated 123,124.

Constitutive over-activation of b-catenin results in increased proliferation of

undifferentiated progenitor cells, with the eventual development of ACTs 123,124.

Activation of the Wnt signaling pathway is commonly seen in adrenocortical neoplasms 125,126. The exact mechanism of Wnt/b-catenin signaling in adrenal cortex function and

development remains unknown 107. However, potential mechanisms include direct

activation of Dax1 by b-catenin 127 and b-catenin induced inhibition of ZF differentiation,

supporting the undifferentiated phenotype of progenitor cells 126,128.

34

1.2.5 Sex Specificity in Adrenal Glands Female adrenal glands are significantly heavier than those of male mice from weeks 3-11,

relative to body weight 129. Additionally, female mice have a significantly larger ZF size

and cell number compared to male mice after 3 weeks of age 129. Both sexes have an X-

zone until approximately week 5 postnatally, when the X-zone starts to recede in male

mice 129. In female mice, the X-zone persists until after the birth of their first litter, when

it will start to recess. However, the role of X-zone and the sex-specificity of this zone is

not fully understood yet 105. Studies investigating corticosterone levels between sexes in a

variety of species including humans have reached different conclusions with some

reporting sex differences in corticosterone levels 78,130, while others found no differences

in corticosterone levels between sexes 129,131-133. Species investigated, time of collection,

method of collection, and age of animal may all be confounding factors, contributing to

the disagreement between the studies. Females are commonly shown to have a higher

corticosterone response to stress, which is also sustained longer than it is in males 78.

Additionally, female mice have a greater number of lipid droplets stored in the adrenal

glands than their male counterparts 129, which indicates a potential for different

steroidogenic activity and capabilities. Additionally, levels of plasma corticosteroid

binding globulin (CBG) vary between sexes, due to role of estrogen in promoting

synthesis of CBG 78. After puberty females are reported to have 2 to 5-fold higher CBG

levels than males 78. Thus, there are numerous sex differences in the growth and

development in the adrenal gland, but the exact mechanism behind these differences

remains largely unknown.

1.3 Adrenal Steroidogenesis

1.3.1 Steroidogenic Pathway Steroidogenesis is the synthesis of all steroid hormones by a variety of P450 enzymes and

hydroxysteroid dehydrogenases, generally located in the adrenal glands, placenta, and

reproductive organs 69. However, low levels of steroidogenesis have been reported in

other tissues134, such as the nervous system 135, skin136, heart 137, and lungs 138. The

steroid hormones produced in each organ is dependent on the specific steroidogenic

enzymes expressed in that organ 69.

35

The initial step of adrenal steroidogenesis begins in the adrenal cortex, where cholesterol

is necessary to produce steroid hormones (Figure 1.7). Most cholesterol for adrenal

steroidogenesis originates from either high-density lipoprotein (HDL) or LDL in the

blood and transport of cholesterol into the cell is mediated by SRB1 receptors for HDL or

LDL receptors for LDL 77. Humans preferentially utilize cholesterol from LDL

endocytosis, while rodents use cholesterol transported by SRB1 receptors 77. Additional

free cholesterol can be produced from de novo synthesis, primarily from the endoplasmic

reticulum 77. Cholesterol in endosomes can be converted into free cholesterol by

lysosomal acid lipase (LAL) 77,139. Free cholesterol can be released from cholesterol

esters stored in lipid droplets by hormone sensitive lipase (HSL) 77,139. Re-esterified

excess free cholesterol by acyl-coenzyme-A-cholesterol-acyl-transferase (ACAT) can be

stored in lipid droplets for future use 77.

36

Figure 1.7: Cholesterol transport for adrenal steroidogenesis.

Free cholesterol for adrenal steroidogenesis is generated by 4 sources. (1) de novo

synthesis from the endoplasmic reticulum; (2) LDL binding to the LDL receptor, which is

taken up by endocytosis into lysosomes, where it will be synthesized from cholesterol

esters to free cholesterol by LAL; (3) HDL cholesterol will bind to SRB1, which can be

immediately used for steroidogenesis or stored in lipid droplets; or (4) HSL provides free

cholesterol from lipid droplets. Excess cholesterol can be stored in lipid droplets after

esterification by ACAT. Abbreviations: LDL, low-density lipoprotein; LAL, lysosomal

acid lipase; HDL, high-density lipoprotein; SRB1, Scavenger receptor B type 1; HSL,

hormone sensitive lipase; ACAT, Acyl-coenzyme-A-cholesterol-acyl-transferase.

37

Within the adrenal cortex cells, steroidogenesis begins within the mitochondria. The rate-

limiting step of adrenal steroidogenesis is the transport of free cholesterol from the outer

mitochondrial membrane (OMM) to the inner mitochondria membrane (IMM), which is

facilitated by the protein StAR (Figure 1.8) 140. Once cholesterol is in the mitochondria,

it can be converted to pregnenolone by the P450 enzyme, cyp11A1 (formally referred to

as P450 side chain cleavage; P450scc). Cyp11A1 conversion is a process of 3 reactions

(1) 22-hydroxylation of cholesterol; (2) 20-hydroxylation of 22(R)-hydroxycholesterol;

and (3) oxidative scission of the C20-22 bond 77,141. Conversion of cholesterol to

pregnenolone is critical for the production of all steroid hormones, so knockout of

cyp11A1 or mutations in this gene result in loss of steroidogenic activity 141. For the

synthesis of glucocorticoids, pregnenolone is converted to progesterone by 3β-

hydroxysteroid dehydrogenase (3β-HSD). In primates that synthesize mainly cortisol but

also low levels of corticosterone, Cyp17 converts pregnenolone to 17a-hydroxylase

pregnenolone and converts progesterone to 17a-hydroxylase progesterone. Progesterone

or 17a-hydroxylase progesterone will then be further converted to 11-

deoxycorticosterone or 11-deoxycortisol by Cyp21 in rodents and humans, respectively 69. Finally, corticosterone/cortisol will be synthesized from 11-deoxycorticosterone/11-

deoxycortisol by an adrenal specific P450 enzyme, Cyp11B1. Corticosterone/cortisol can

either exit the adrenal gland in the plasma, bound to CBG and transported to most target

organs throughout the body 78 or can be further converted by Cyp11B2 to aldosterone in

the ZG of the adrenal cortex 142.

38

Figure 1.8: Steroidogenic pathway involved in glucocorticoid synthesis.

Steroidogenesis starts with the transport of free cholesterol from the outer mitochondria

membrane to the inner mitochondria membrane by StAR, which can then be further

converted to all the major steroid hormones. Abbreviations: StAR, steroidogenic acute

regulatory protein; cholesterol side chain cleavage enzyme, cyp11A1, 3β-HSD, 3β-

hydroxysteroid dehydrogenase; cyp21, 21-hydroxylase; cyp11b1, steroid 11β-

hydroxylase; cyp11b2, aldosterone synthase; cyp17, cytochrome P450 17A1; cyp19,

aromatase; 17β-HSD, 17β-hydroxysteroid dehydrogenases.

39

1.3.2 Steroidogenic Acute Regulatory Protein StAR is the rate-limiting step in steroidogenesis, due to its essential role of transporting

cholesterol from the OMM to the IMM for steroidogenesis. Mutations of StAR in humans

produce congenital lipoid adrenal hyperplasia, resulting in a lack of steroid hormone

production that requires life-long hormone therapy 143. Moreover, StAR knockout mice

lack the ability to synthesize steroid hormones, and accumulate cholesterol in both the

adrenal glands and gonads 144-147. StAR-/- mice have external female genitalia and fail to

grow after birth 147. A proportion of StAR-/- mice die shortly after birth due to respiratory

distress, and the remainder die a week after birth from an imbalance in fluid and

electrolytes, a result of secondary adrenal insufficiency 147. While StAR-/- mice can be

rescued by treatment with steroid hormones (corticosterone and aldosterone), they retain

notable abnormalities in adrenal and gonad structure and function 146. Steroidogenesis is

not completely abrogated in StAR-/- mice until the accumulation of lipid droplets in

adrenal and gonads builds up enough to destroy steroidogenic cells 146.

StAR originates from a 37-kDa StAR pre-protein with an N-terminal mitochondrial

targeting sequence that directs it to the mitochondria 77,141. The 37-kDa StAR cytoplasmic

precursor has a short half-life, and is rapidly degraded if not imported into the

mitochondria (Figure 1.9) 141. Cleavage of the 37-kDa StAR pre-precursor into a 30-kDa

“mature” molecule by removal of the N-terminus occurs at the OMM 148. Although the

30-kDa protein (referred to in this thesis only as StAR) is considered “mature”, the

removal of the N-terminus is not necessary for activation 77. The cleavage, however,

seems to contribute to the localization of StAR on the OMM, which does determine its

activity 77,149. Thus, the time of StAR residency on the OMM is directly proportional to

its activity 77,141. StAR contains a sterol binding pocket allowing it to transport a single

cholesterol molecule from the OMM to the IMM. 150. However, each StAR molecule will

transport hundreds of cholesterol molecules before it undergoes cleavage and removal

from the OMM, terminating its activity 141. There are currently four proposed models to

account for StAR’s ability to transport cholesterol 151. (1) Contact sites: the 37-kDA

StAR forms contact sites with the OMM and IMM that permit cholesterol to flow down a

concentration gradient into the mitochondrial matrix 151,152. (2) Desorption: StAR

“desorbs” cholesterol at the OMM, permitting its entry into the intra-mitochondrial space

40

(IMS), potentially as micro droplets 151,153. There is little evidence to support this theory,

since micro droplets of cholesterol have not been observed 151. (3) IMS Shuttle: StAR acts

in the IMS to shuttle cholesterol from the OMM to the IMM 151,154. This theory is no

longer accepted, due to the observation of StAR activity on the OMM 151. (4) Molten

Globule: StAR undergoes a conformational change at the OMM caused by protonated

phospholipids 149,151. This is confirmed by the dependence of StAR on a proton pump on

the mitochondria for its activity 151,155. Additionally, at the OMM StAR interacts with a

multi-protein complex containing translocation protein (TSPO, previously the peripheral

benzodiazepine receptor; PBR), voltage-dependent anion channel 1, and phosphate

carrier protein, which may all be involved in StAR-mediated cholesterol transport 148,149,156,157. The activity of steroidogenesis is partially controlled by the phosphorylation

of StAR at Ser194/5, which doubles its rate of cholesterol transport 77,158. In the absence

of StAR, steroidogenesis is possible when cholesterol transport occurs with the help of

metastatic lymph node 64 protein (MLN64) using a yet to be determined mechanism 159.

This MLN64 mediated process of cholesterol transport occurs mostly in the human

placenta, which lacks StAR protein, and has about 50-60% of the cholesterol transport

ability of StAR 159.

41

Figure 1.9: Synthesis of steroidogenic acute regulatory protein (StAR).

(1) Transcription of StAR occurs in the nucleus of steroidogenic cells. (2) StAR mRNA

is then transported to the mitochondria where it binds to AKAP149, which is involved in

its translation into a 37-kDa pre-protein and phosphorylated by PKA at Ser194/5. (3) 37-

kDa StAR will then interact with the multi-protein complex, TSPO on the outer

mitochondria membrane, where the n-terminus of StAR is cleaved. StAR then facilitates

the transport of cholesterol from the outer mitochondria membrane to the inner

mitochondria membrane. (4) After cholesterol transport, 30-kDa StAR will enter the

mitochondria where it will be degraded. Abbreviations: StAR, steroidogenic acute

regulatory protein; AKAP149, A-kinase anchor proteins; PKA, protein kinase A; TSPO,

Translocator protein.

42

1.3.2.1 Regulation of StAR Due to the essential role of StAR in facilitating cholesterol transport from the OMM to

the IMM for steroidogenesis, the regulation of this protein is of great importance. The

regulation of StAR is not fully understood but has been shown to be quite complex,

involving numerous hormones, transcription factors, and receptors 160,161.

1.3.2.1.1 Epigenetic Regulation of StAR StAR expression is affected directly and indirectly by epigenetic modifications, such as

histone modifications, and micro-RNA (miRNA) 162-166. For example, induction of StAR

was associated with acetylation of histone 3 but not histone 4 on the proximal StAR

promoter in MA-10 cells and in primate granulosa cells after stimulation 162,163.

Additionally, epigenetic factors can have indirect effects on StAR expression by altering

transcription factors known to bind the StAR promoter, such as miRNA-133b inhibition

of the negative transcription factor forkhead box L2 (Foxl2), which results in increased

StAR transcription 164.

1.3.2.1.2 Transcriptional Regulation of StAR The first 150 bases of the proximal StAR promoter are highly regulated by numerous

transcription factors. Positive regulators of StAR transcription include SF-1, CCAAT-

enhancer-binding protein β (C/EBPβ), GATA-4, specificity protein 1 (Sp1), sterol

regulatory element binding protein (SREBP), CREB/CREM, and AP-1 140,162,167, all of

which have numerous putative binding sites on the StAR promoter 140,167. Additionally,

DAX-1, Foxl2 and Yin Yang 1 (YY1) negatively regulate transcription of StAR mRNA.

Transcription factor expression and regulation of StAR have been shown to be cell/tissue-

specific as well as time dependent 162.

SF-1 not only plays a critical role in adrenal development, it is also known as a master

regulator of steroidogenesis 168. There are six binding sites for SF-1 on the StAR

promoter, where it can regulate both basal and stimulated StAR transcription 140,167. The

SF-1 binding sites -43/-37, -102/-96 and -105/-99 have all been shown to be essential for

both basal and stimulated StAR regulation in reproductive cells 167. Mutations in any SF-

1 binding sites, result in a significant decrease, but not an elimination in cAMP induced

43

StAR activity, demonstrating the involvement of other transcription factors or regulatory

responses in StAR expression.

These transcription factors are able to act alone or in combination by binding to the StAR

promoter 167. It is suggested that C/EBPβ can form a complex with SF-1 on the StAR

promoter, as well as interacting with Sp1 to promote StAR transcription 140,167.

Additionally, SF-1 can interact with Sp1, and C/EBPβ cooperates with GATA-4 to

regulate StAR expression, 167. Therefore, the regulation of StAR transcription is thought

to involve the interaction of numerous transcription factors at the StAR promoter region.

1.3.2.1.3 Post-transcriptional Regulation of StAR Initially, StAR was thought to be mainly transcriptionally regulated, however recent

evidence points to a role of post-transcriptional regulation of StAR 160. The stability of

StAR mRNA and post-translational regulation of StAR have been investigated 160.

Currently, no known proteins have been shown to bind to StAR mRNA to regulate its

stability, however, possible candidates include the mRNA stabilizing proteins, TPA-

induced sequence 11b (Tis11b) and HuR, which are expressed in steroidogenic cells 160.

Additionally, StAR mRNA may be targeted by proteins such as mevalonate kinase,

DAX-1 and A-kinase anchoring protein 121/149 (AKAP121/149) that alter its rate of

translation 160. AKAP121/149 contains an N-terminal KH domain that targets and recruits

StAR mRNA to its location at the OMM where it can be translated 169. StAR protein

expression at the OMM has been shown to be enhanced by AKAP121/149 in MA-10

cells 170. In addition, AKAP121/149 has been demonstrated to recruit PKA, which

phosphorylates StAR to increase its activity 170.

The N-terminal of the 37-kDa StAR directs it to the mitochondria, but it may also

destabilize the protein, promoting its degradation and contributing to its short half-life 152,160. StAR degradation in the cytoplasm has been shown to be extremely rapid in many

tissues 171. Proteasome-mediated degradation of the 37-kDa StAR protein has been

demonstrated, and it is suggested that this may occur without ubiquitinylation of StAR 171.

StAR physically and/or functionally interacts with numerous proteins at or around the

OMM, including cyclin dependent kinase-5 (CDK5), mitochondrial kinases (MEK) 1/2,

44

PAP7, TSPO, HSL 160. While these proteins and others could potentially contribute to

StAR’s expression and activity, more evidence is needed to understand StAR–protein

interactions.

1.4 Effects of BPA on Steroidogenesis

1.4.1 Male Reproductive Steroidogenesis Due to its estrogenic nature, the effect of BPA on the male reproductive system has been

the subject of considerable research effort. BPA exposure, both during the prenatal period

and during adulthood results in increased prostate size 6,172. Rodent studies have

demonstrated that exposure to BPA during the developmental period alters

spermatogenesis and reduces sperm quality in adult offspring 173. Additionally, BPA

adversely affects androgen production, which is essential for functional spermatogenesis 173. Decreases in testosterone levels and/or steroidogenic enzymes was observed in

rodents exposed to BPA prenatally, as well as during the postnatal period 173-176.

Investigation of the effects of BPA on the rate limiting step of steroidogenesis, StAR, in

the male reproductive system yielded inconsistent results that vary between model

systems (Table 1.3 and 1.4). In male rats, Qui et al. 2003 177 observed an increase in

StAR and cyp11A1 gene expression in the testis after acute BPA exposure. In contrast,

D'Cruz, et al. 175 demonstrated that BPA exposure resulted in decreased protein

expression of StAR in male rats, a finding in agreement with two other studies showing

that acute BPA exposure decreased StAR levels in testes 174,178. Chouhan, et al. 178

concluded that the decrease in StAR protein after BPA exposure could be attributed to a

BPA-induced increase in oxidative stress, resulting in increased inducible nitric oxidative

synthase (iNOS). Moreover, both perinatal and acute exposure to BPA significantly

inhibited StAR in the testis of fetal and offspring rodents 179-181. However, BPA treatment

for 17 h did not significantly alter levels of StAR in primary mouse Leydig cells 182.

Therefore, more research is needed to understand the variability and mechanism behind

the effects of BPA on StAR in the male reproductive system.

1.4.2 Female Reproductive Steroidogenesis Due to BPA activity as an estrogen mimicking chemical, the effects of BPA on the

female reproductive system and fertility has also been investigated. Of interest is a study

45

by Ikezuki, et al. 59 that used enzyme-linked immunosorbent assays (ELISA) to determine

BPA levels of 1-2 ng/mL in 36 human follicular fluid samples. In contrast, when

measuring BPA with high-performance liquid chromatography and mass spectrometry

(HPLC-MS), no BPA was detected in the five human follicular samples examined 183.

Ovarian steroidogenesis is fundamental for estrogen production, which is essential for

ovarian function 184. In ovaries, theca cells use cholesterol to produce testosterone via the

steroidogenic pathway, which is then further converted to estrogen in granulosa cells by

the enzyme aromatase (cyp19A) 184. The effects of BPA on estrogen, androstenedione

and DHEA have been widely reported in the literature 184. In vitro studies have

demonstrated increased estrogen synthesis, and as shown by Peretz, et al. 185, impairment

of follicular growth. However, BPA exposure in rodent models result in inconsistent

outcomes184. Gamez, et al. 186 reported increased estradiol and FSH levels in pre-pubertal

female rats exposed to 3µg/kg/d BPA prenatally. In contrast, ovine female offspring

prenatally exposed to three different BPA doses (0.05, 0.5, or 5mg/kg bw/day) had no

changes in estradiol levels, but did have a shortened time of estradiol surge compared to

the LH surge peak, indicating potential fertility problems 187. Epidemiological studies

have also found that higher BPA levels lead to higher serum estradiol levels in most cases 184. However, few human studies employ healthy female subjects, tending to focus on

women attending clinics for in vitro fertilization or other reproductive conditions 184.

Thus, the effects of BPA on ovarian steroidogenesis and function appear to be dose-,

species- and time-dependent, with more investigation necessary to provide conclusive

results.

Numerous investigators have looked at levels of the key steroidogenic protein StAR,

since it is involved in all steroid hormone production as well as being the rate-limiting

step in steroidogenesis (Table 1.3 and 1.4). BPA inhibits StAR in cultured mouse

ovarian follicles in a variety of mouse strains 185,188,189. Conversely, BPA increases

cyp11A1 and StAR mRNA in rat ovarian theca-interstitial (T-I) cells and granulosa cells 190. However, BPA had no effect on StAR mRNA in luteinized human granulosa cells 191.

In vitro studies showed that BPA inhibited StAR protein expression in T-I and granulosa

cells 192. In contrast, Xi, et al. 179 found no change in ovarian StAR expression with either

prenatal or postnatal BPA exposure. Thus, the effects of BPA exposure on steroidogenic

46

enzymes are animal- and tissue-specific and vary depending on the type of BPA

exposure. Nevertheless, it is important to note that regulation of steroidogenesis in

reproductive tissues differs from that of the adrenal gland.

47

Table 1.3: Effects of BPA on testicular and ovarian StAR expression in vitro.

Cell type Time of BPA Dose Results Reference

Primary antral follicles from FVB, and C57BL/6

24-96 h 1-100 µg/ml Decreases mRNA of StAR after 72-96 h after 10-100 µg/ml BPA.

189

Primary CD-1 mouse antral follicles

24-96 h 1-100 µg/ml Decreases mRNA of StAR at 72-96 h with 10-100 µg/ml

188

Primary mouse follicles from FVB mice

120 h 4.4-440 µM Decreases StAR after 440 µM

185

Primary rat theca-interstitial and granulosa cells

72 h 10-7-10-4 M Increases StAR mRNA after 10-5-10-4 M in theca cell and 10-4 M in granulosa cells

190

Luteinized human granulosa cells

48 h 0.02, 0.2, 2, 20 µg/ml

No effect on StAR mRNA

191

Primary culture of mouse leydig cells

17 h 10 µM No effect on StAR mRNA

182

48

Table 1.4: Effects of BPA on testicular and ovarian StAR expression in vivo.

Animal Model Tissue Time of

exposure Dose and method Results Reference

Swiss albino male mice

Testes 60 days IP injection of 0.5, 50 and 100 µg/kg body weight/day

Decrease StAR protein at all doses

178

Wistar/ST male rats

Testes 42 days SC injections of 20, 100, or 200 mg BPA/kg/day

StAR mRNA and protein decreased with 100 and 200 mg dose

174

Wistar male rats

Testes 45 days Gavaged 0.005, 0.5, 50, and 500 µg/kg bw/day

Decreased StAR protein at all doses

175

Sprague Dawley male Rats

Testes 56 days Gavaged 0.0005, 0.5, 5 mg/kg/bw

Increases StAR mRNA and protein at 5mg/kg/bw dose

177

Sprague Dawley female rats

Ovary 90 days Gavaged with 0.001, or 0.1 BPA mg/kg bw

Decrease StAR protein at all doses

192

Sprague-Dawley rats

Fetal testes

E11-20 SC injections of 0.02, 0.5, or 400 mg/kg/day

Decreased StAR mRNA at 400 mg/kg/day

180

ICR Mice Offspring adult testes

E1-5 20 µg/kg/day orally

StAR mRNA decreased at PND 35-50

181

CD-1 mice Ovary and testes

Cohort A: E1-PND 49 Cohort B: PND 20-49

Gavaged Cohort A: 12-50 mg/kg/day Cohort B: 25-50 mg/kg/day

Cohort A: decreased StAR mRNA and protein at 50mg/kg/day in testes, no change in ovaries Cohort B: no change in StAR mRNA

179

49

1.4.3 Adrenal Steroidogenesis Due to the essential role of glucocorticoid in maintaining whole body homeostasis,

epidemiological studies have associated high levels of BPA with HPA dysfunction 57,193.

A recent study by Giesbrecht, et al. 57 demonstrated that pregnant women with high

urinary BPA (1.66-43.20 ng/mL) had lower waking cortisol levels and flatter diurnal

cortisol rhythms, providing evidence for the potential of BPA to alter the HPA axis and

cortisol response in adults 57. The offspring of the same women were examined after

parturition, to determine the effects of high BPA exposure on infant cortisol levels and

reactivity 193. This study showed an association between high maternal BPA levels and

increased basal salivary cortisol levels in female infants, but decreased cortisol levels in

male infants compared to infants exposed to low BPA levels 193. Additionally, cortisol

reactivity was decreased in female infants and increased in male infants exposed to high

prenatal BPA 193. Taken together, these studies shown an association of chronic prenatal

exposure to BPA with dysfunction of the HPA-axis in humans 57,193.

The effects of BPA on plasma levels of corticosterone have been evaluated in numerous

experimental animal studies, however the effects appear to be dependent on the animal

used, dosage of BPA, timing and length of exposure, route of exposure, and time of

corticosterone measurement (Table 1.5). The sex-specificity of BPA effects on

corticosterone levels remain disputed. An increase in corticosterone levels in male but not

female adult offspring was seen in rats pre- and post-natally exposed to 2 µg/kg

subcutaneous injections of BPA from E10 to PND7 80,81. However, an increase in

corticosterone levels was seen in female, but not male, mid-adolescence rat offspring

when pre- and post-natally exposed to 40 µg/kg BPA in orally throughout pregnancy and

lactation 194,195. No changes in corticosterone were seen in either sex of rats at PND21

when gavaged with 2.5 or 25 µg/kg/day BPA from E6 to PND21 196.

The effects of acute BPA exposure on adrenal steroidogenic enzymes have been

demonstrated in the adrenal mouse cell line Y-1, as well as in rats acutely exposed to

BPA 197. Lan et al. 197 demonstrated that in vitro exposure to BPA levels from 50-10,000

nM was sufficient to elevate cyp11A1 protein levels in a dose-dependent manner, but did

not affect SF-1 levels 197. Additionally, this group showed that daily subcutaneous BPA

injections of 0.5 µg/kg for three days resulted in increased plasma corticosterone and

50

adrenal cyp11A1 protein levels in male Sprague-Dawley rats 197. These studies show that

BPA alters adrenal steroidogenesis in cell and animal models; however, the effects of

prenatal BPA exposure on adrenal steroidogenesis have yet to be investigated.

51

Table 1.5: Effects of BPA on basal corticosterone levels.

Animal Model

Time of exposure

Dose and Method

Age of evaluation

Basal Corticosterone

levels Reference

C57BL/6 E7.5-E18.5 25 mg

BPA/kg in food

E18.5 No change 68

Sprague-Dawley rats E10-PND7

orally administered 2 µg/(kg/day)

of BPA

PND80 Increased in males

81

Sprague-Dawley rats

E6-E21 prenatally,

PND1-PND21

directly to pup

Orally gavaged 2.5

and 25 µg/kg/day

PND21 No change in either sex

196

Sprague-Dawley rats

Throughout pregnancy

and lactation

orally gavaged

40 µg/kg/day of BPA

PND40-50 Increased in females

198

Wistar rats

Throughout pregnancy

and lactation

orally administered 40 µg/kg/day

of BPA

PND46 Increased in

females and not males

195

Wistar rats

Throughout pregnancy

and lactation

orally administered 40 µg/kg/day

of BPA

PND46 Increased in

females but not males

194

Sprague-Dawley rats E10-PND7

2µg/(kg/day) BPA

SC injections PND80

Increased in males and not

females 80

Deer mice

2 weeks prior to

mating and throughout pregnancy

and lactation

50mg of BPA/kg feed

weight PND90 No changes in

males 199

Sprague-Dawley rats 3 days

0.5µg/kg BW BPA

SC injections 8 weeks Increased in

males 197

52

1.5 Rationale BPA as an EDC in numerous tissues is well established 2,10,15. Moreover, the potential

adverse effects of in utero BPA exposure on fetal development and the long-term

consequences of this exposure is of great concern 2,6,13. Indeed, prenatal BPA exposure

has a wide range of adverse health effects, including reproductive 179-181, cardiovascular 200,201, respiratory 68,202, immunological 203,204, metabolic 66,67,205, behavioral and

neurological 11 disorders, in both the fetus and adult offspring. Thus, brief exposure to

BPA during critical periods of development can have lifelong health consequences.

The adrenal gland plays a critical role in production of glucocorticoids which are

necessary in maintaining whole body homeostasis. Furthermore, the adrenal gland is

highly vulnerable to environmental toxin insult due in part to its potential for free radical

generation during steroidogenesis, ability to take up lipophilic agents, high vascularity

allowing delivery of toxins, and high levels of CYP enzymes available to activate toxins 206,207. Given the above, my thesis focuses on the long-term effects of prenatal BPA

exposure on adrenal gland development and steroidogenic function in adulthood.

Prenatal BPA exposure has been shown to increase plasma glucocorticoid levels in

offspring, in a sex-specific manner. However, the precise nature of these sex-specific

effects on plasma corticosterone levels remains obscure 80,194,195,198. Moreover, whether

the BPA-induced increases in plasma corticosterone levels are a result of enhanced

adrenal steroidogenesis is not known. Importantly, whether prenatal BPA exposure alters

adrenal development remains to be demonstrated. Therefore, this thesis addresses these

important questions.

1.6 Hypothesis I hypothesize that prenatal exposure to BPA disrupts adrenal gland development and

steroidogenic function in adult mouse offspring.

1.7 Objectives i. To determine the effects of prenatal BPA exposure on adrenal gland development,

and adrenal steroidogenic function in vivo.

53

ii. To determine the molecular mechanisms that underlie the BPA-induced aberrant

adrenal gene expression in vitro.

iii. To determine the molecular mechanisms underlying the BPA-induced aberrant

adrenal gland development in vitro.

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2 PRENATAL EXPOSURE TO BISPHENOL A DISRUPTS STEROIDOGENESIS IN ADULT MOUSE OFFSPRING1

1 Reproduced (adapted) from: Medwid S, Guan H, Yang K (2016) Prenatal exposure to bisphenol A disrupts steroidogenesis in adult mouse offspring. Environ Toxicol Pharmacol. 43: 203-8

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2.1 Introduction Bisphenol A (BPA) is a ubiquitous endocrine disrupting chemical, being present in

polycarbonate plastics, epoxy resins, paper receipts, and cardboards, as well as water and

air samples 1-3. Of foremost concern is exposure to BPA during the critical period of

organ maturation 4. Indeed, BPA has been detected in placental tissues and fetal blood,

demonstrating BPA’s ability to cross the placenta and reach the fetus 3,5. Numerous

human epidemiological studies have demonstrated an association between gestational

exposure to BPA and pregnancy complications, male genital abnormalities, childhood

obesity, childhood asthma and altered neurological development in children 6.

Furthermore, in vivo animal studies have shown that developmental BPA exposure results

in a wide range of adverse effects, including reproductive, cardiovascular,

immunological, metabolic, behavioural, and neurological disorders as well as certain

cancers in adult offspring. In addition, many of these adverse effects are sex specific 3,4,7.

Due to the critical role of glucocorticoids (cortisol in humans, and corticosterone in

rodents) in maintaining whole body homeostasis, the effects of BPA on the

hypothalamic-pituitary-adrenal axis had been examined previously. It was found that

maternal exposure to BPA during pregnancy and lactation resulted in increased basal

corticosterone levels in juvenile female, but not male rats 8-10. Furthermore, perinatal

BPA exposure led to abnormal adrenal cortex structure, including increased adrenal gland

weight in females, accompanied by a reduction in the zona reticularis and hyperplasia of

the zona fasciculata in both sexes 8. In contrast, Chen, et al. 11 reported that prenatal BPA

exposure resulted in increased corticosterone levels in adult male rats only. Thus, the

precise nature of these sex-specific effects of developmental exposure to BPA on

circulating corticosterone levels remains obscure. Importantly, whether the BPA-induced

increases in circulating corticosterone levels are a result of enhanced adrenal

steroidogenesis is unknown. Therefore, the present study was undertaken to address these

two important questions.

74

2.2 Materials and Methods

2.2.1 Animal Experiments The use of animals in this study was approved by the Council on Animal Care at the

University of Western Ontario, following the guidelines of the Canadian Council on

Animal Care. Breeding pairs of adult C57BL/6 mice were purchased from Charles River

Laboratories (Wilmington, MA). To minimize environmental BPA exposure, mice were

housed in polypropylene cages with glass water bottles. Mice were allowed food and

water ad libitum, and maintained in humidity and temperature-controlled rooms on a

12 h/12 h light-dark cycle. Female mice were placed overnight with males, and detection

of vaginal plug the next morning indicated pregnancy, and marked as embryonic day 0.5

(E0.5). Pregnant dams were fed either a control diet (phytoestrogen-free food pellets

supplemented with 7% corn oil; TD.120465, Harlan Teklad, Madison, WI) or the control

diet supplemented with 25 mg BPA/kg feed weight (equivalent to 5 mg BPA/kg body

weight; TD.120466, Harlan Teklad, Madison, WI) from E7.5 to postnatal day (PND) 0.5.

The gestational age of E7.5 was chosen as the start of the feeding regime in order to

avoid any confounding effects of BPA on embryo implantation. After birth, both control-

and BPA-fed dams were switched to regular chow for remainder of the study. Pups were

weaned on PND 21, separated by sex and fed regular chow. Five litters of control and

BPA were used, with number of pups between 6 and 11 pups per litter. Litters were

culled at 5 pups per sex per litter. They were group housed by litter, experimental

treatment and sex. Offspring were sacrificed between 8 and 10 weeks of age using carbon

dioxide asphyxiation. Blood samples were collected via cardiac puncture in heparinized

capillary tubes (Fisher brand Cat. No. 22-260-950), and centrifuged at 2000g for 10 min

at 4 °C. Plasma was then harvested were stored at −80 °C. Adrenal glands were dissected,

weighed, snap-frozen in liquid nitrogen, and stored at −80 °C. All sacrifices and sample

collection were done between 9:00–11:00 am.

2.2.2 Western Blot Analysis Due to the limited tissue quantity (i.e., the tiny size of mouse adrenal glands), we made a

strategic decision to determine changes in protein, rather than mRNA, abundance

following exposure to BPA during critical periods of adrenal gland development. This is

because information on alterations in protein levels is more biological meaningful.

75

Furthermore, the limited tissue availability also precluded the possibility of conducting

enzyme activity assays, which would require greater amounts of tissues in comparison to

western blot analysis.

Levels of various proteins were analyzed using standard western blot analysis, as

previously described 12. Briefly, sodium phosphate buffer, (pH 7.0), was used at a 10

times volume dilution to hand homogenize 2–3 adrenals gland from the same sex and the

same litter before being mixed with equal amounts of SDS gel loading buffer (50 mM

Tris-HCL, pH 6.8, 2% wt/vol SDS, 10% vol/vol glycerol, 100 mM DTT and 0.1% wt/vol

bromophenol blue) to be loaded to a standard 10% SDS-PAGE gel. Protein was then

transferred to a PVDF transfer membrane (Amersham Hybond-P, Cat. No. RPN303F, GE

Healthcare Lifesciences, Baie D'Urfe, QC), and blocked overnight with 5% milk in TTBS

(0.1% vol/vol Tween-20 in TBS) to decrease non-specific antibody binding. Membranes

were then probed with primary antibodies (Table 2.1) for 1–2 h at room temperature.

Washing was done with TTBS, 3 × 10 min before labeling with horseradish peroxidase-

labeled rabbit secondary antibody (Table 2.1), for 1 h at room temperature. After

3 × 10 min TTBS washes, proteins were detected using ECL and visualized using a

chemiliminescence (Cat. No. WBLUR0500, Luminata Crescendo, Western HRP

Substrate; Millipore, Etobicoke, ON) and captured on the VersaDoc Imaging System

(BioRad, UK). Densitometry was performed using Image Lab Software, comparing

levels of proteins expressed as percent of controls.

76

Table 2.1: Primary and secondary antibodies used for western blotting.

Antibody Company Catalog Number Dilution Used

Perilipin Cell Signaling 3470 1:1000

StAR Santa Cruz Sc-25806 1:4000

Cyp11A1 Bioss Bs-3608R 1:200

SF-1 Abcam Ab168380 1:1000

GAPDH Imgenex IMG-5567 1:10000

Anti-Rabbit R&D systems HAF008 1:3000

77

2.2.3 Hormone Assays Levels of corticosterone and ACTH in plasma samples (plasma from one litter and the

same sex were pooled, and used as one sample) were determined with an ELISA Kit

following the manufacturer’s instructions (corticosterone: Abcam, ab108821, 1:60

plasma dilution, Toronto, ON; ACTH: Phoenix Peptide EKE-001-21, 1:1 plasma dilution,

Burlingame, CA). To eliminate inter-assay variations, all samples were analyzed in

triplicate in one assay, and the intra-assay coefficient of variation was <5%.

2.2.4 Statistical Analysis Results are presented as mean ± SEM of four to five different litters, as indicated in

figure legends. Data were analyzed using two-way ANOVA followed by Tukey’s post-

hoc test, or Student’s t-test as indicated. Significance was set at p < 0.05. Calculations

were performed using Graphpad Software Prism version 6.

2.3 Results

2.3.1 Effects of prenatal BPA exposure on adrenal gland weight To determine if prenatal BPA exposure affected body weight, mice were weighed at 8

weeks. No significant differences in body weight were observed in either sex of BPA-

exposed and non-exposed control mice (Figure 2.1A & B). To investigate if prenatal

BPA exposure resulted in altered adrenal gland weight, the weight of adrenal glands and

the ratio of adrenal gland weight to body weight were determined. An increase in adrenal

gland weight was observed in both male (P < 0.05) and female (P < 0.01) mice prenatally

exposed to BPA when compared with controls (Figure 2.1C & D). Furthermore, the ratio

of adrenal gland weight to body weight was significantly increased in both BPA-exposed

male (P < 0.05) and female (P < 0.01) mice (Figure 2.1E & F).

78

Pregnant mice were fed a control diet (phytoestrogen free food pellets) or the control diet

supplemented with 25 mg BPA/kg food pellets from E7.5 to birth. At eight weeks of age,

offspring were sacrificed, body weight (A & B) and adrenal gland weight (C & D) were

recorded, and adrenal gland to body weight ratio (E & F) was then calculated. Data are

presented as mean ± SEM (n = 16–22; *P < 0.05, **P < 0.01, vs. control).

C BPA0.0

0.1

0.2

0.3

0.4**

Adre

nal w

eigh

t/BW

C BPA0

2

4

6

8 **

Adre

nal G

land

W

eigh

t (m

g)

C BPA0

10

20

30

Body

Wei

ght (

g)

A

C

B

D

E F

Female Male

C BPA0

10

20

30

Body

Wei

ght (

g)

C BPA0

2

4

6

8

*Ad

rena

l Gla

nd

Wei

ght (

mg)

C BPA0.0

0.1

0.2

0.3

0.4

*

Adre

nal w

eigh

t/BW

Figure 2.1: Effects of prenatal BPA exposure on adrenal gland weight.

79

2.3.2 Effects of prenatal BPA exposure on basal plasma corticosterone and ACTH levels

To determine if prenatal BPA exposure affected adrenal plasma corticosterone, plasma

corticosterone levels were measured using ELISA. We found that corticosterone levels

were significantly increased in both male (P < 0.01) and female (P < 0.05) mice

prenatally exposed to BPA when compared to control mice (Figure 2.2A). To ascertain if

elevated corticosterone levels were a result of hyper-pituitary activity, plasma ACTH

levels were measured with ELISA. We observed no differences in plasma ACTH levels

between control and prenatally BPA-exposed mice (Figure 2.2B).

80

Pregnant mice were fed a control diet (phytoestrogen free food pellets) or the control diet

supplemented with 25 mg BPA/kg food pellets from E7.5 to birth. At eight weeks of age,

offspring were sacrificed, and plasma samples were collected. Plasma levels of

corticosterone (A) and ACTH (B) were measured by standard ELISA. Data are presented

as mean ± SEM; statistical significance was determined using a 2-way ANOVA followed

by Tukey’s post-hoc test (n = 3–5; *P < 0.05, **P < 0.01, ***P < 0.001).

C BPA C BPA0.0

0.5

1.0

1.5

Male Female

AC

TH (n

g/m

l)

A B

C BPA C BPA0

250

500

750

1000 ****

Male Female

***

Cor

ticos

tero

ne

(ng/

ml)

**

Figure 2.2: Effects of prenatal BPA exposure on plasma corticosterone and ACTH

levels.

81

2.3.3 Effects of prenatal BPA exposure on perilipin protein levels To determine if prenatal exposure to BPA altered substrate availability for adrenal

steroidogenesis, adrenal levels of perilipin, a surrogate for cholesterol content 13, were

measured using western blot analysis. The level of perilipin protein was similar between

control and prenatally BPA-exposed mice in both sexes (Figure 2.3).

82

Figure 2.3: Effects of prenatal BPA exposure on perilipin protein levels in adrenal

glands.

Pregnant mice were fed a control diet (phytoestrogen free food pellets) or the control diet

supplemented with 25 mg BPA/kg food pellets from E7.5 to birth. At eight weeks of age,

offspring were sacrificed, and adrenal glands were collected. Levels of perilipin protein

in adrenal gland tissue homogenates were determined separately in males (A & C) and

females (B & D) by western blot analysis. Data are presented as mean ± SEM (n = 4).

Perilipin GAPDH

C D

Control BPA Females Males

Control BPA A B

C BPA 0

50

100

150Pe

rilip

in/G

APD

H (%

of c

ontro

l)

C BPA 0

50

100

150

Peril

ipin

/GAP

DH

(% o

f con

trol)

37kDa

62kDa

83

2.3.4 Effects of prenatal BPA exposure on StAR and cyp11A1 protein levels

To study the effects of prenatal exposure to BPA on the rate-limiting steps of

steroidogenesis, levels of StAR and cyp11A1 proteins were measured by western

blotting. We found a significant increase in both StAR (P < 0.01) and cyp11A1 (P < 0.05)

protein levels in prenatally BPA exposed female mice compared to controls (Figure

2.4B, D & F). By contrast, no changes in either StAR or cyp11A1 protein were observed

in male mice prenatally exposed to BPA when compared to control males (Figure 2.4A,

C & E).

84

Figure 2.4: Effects of prenatal BPA exposure on StAR and Cyp11A1 protein levels.

Pregnant mice were fed a control diet (phytoestrogen free food pellets) or the control diet

supplemented with 25 mg BPA/kg food pellets from E7.5 to birth. At eight weeks of age,

offspring were sacrificed, and adrenal glands were collected. Levels of StAR and

cyp11A1 protein in adrenal gland tissue homogenates were determined separately in

males (A, C & E) and females (B, D, & F) by western blot analysis. Data are presented as

mean ± SEM (n = 4; *P < 0.05, **P < 0.01 vs. control).

cyp11A1 GAPDH

StAR

C D

E F

Control BPA Males Females

BPA Control A B

C BPA 0

50

100

150

200St

AR/G

APD

H(%

of c

ontro

l)

C BPA 0

50

100

150

200

cyp1

1A1/

GAP

DH

(% o

f con

trol)

C BPA0

50

100

150

200**

StAR

/GAP

DH

(% o

f con

trol)

C BPA0

50

100

150

200*

cyp1

1A1/

GAP

DH

(%

of c

ontro

l)

37kDa

30kDa 53kDa

85

2.3.5 Effects of prenatal BPA exposure on SF-1 protein levels To investigate the effects of prenatal exposure to BPA on the regulatory mechanisms of

adrenal steroidogenesis, we examined the expression of steroidogenic factor-1 (SF-1), a

key transcription factor involved in the regulation of StAR and cyp11A1. We found no

significant changes in the level of SF-1 protein in either female or male mice prenatally

exposed to BPA when compared to control mice of the same sex (Figure 2.5).

86

Figure 2.5: Effects of prenatal BPA exposure on SF-1 protein levels in adrenal

glands.

Pregnant mice were fed a control diet (phytoestrogen free food pellets) or the control diet

supplemented with 25 mg BPA/kg food pellets from E7.5 to birth. At eight weeks of age,

offspring were sacrificed, and adrenal glands were collected. Levels of SF-1 protein in

adrenal gland tissue homogenates were determined separately in males (A & C) and

females (B & D) by western blot analysis. Data are presented as mean ± SEM (n = 4).

SF-1 GAPDH

C D

Control BPA Males Females

Control BPA A B

C BPA 0

70

140

210

280

SF-1

/GAP

DH

(%co

ntro

l)

C BPA0

70

140

210

280

SF-1

/GAP

DH

(% o

f con

trol)

37kDa 53kDa

87

2.4 Discussion In the present study, we demonstrate that prenatal exposure to BPA results in increased

basal corticosterone levels independent of circulating ACTH levels in both male and

female adult mouse offspring. Furthermore, we provide evidence indicating that BPA-

induced increases in basal corticosterone levels are likely a consequence of up-regulated

adrenal steroidogenesis in female mice while the mechanisms behind the BPA-induced

increase in corticosterone levels in males are unknown. Thus, our present findings

provide novel insight into the long-term and sex-specific effects of developmental BPA

exposure on adrenal steroidogenesis.

The dose of BPA used in this study (25 mg BPA/kg diet; equivalent to 5 mg BPA/kg

body weight) was chosen based on our previous dose-response studies in which we found

that prenatal exposure to this dose of BPA led to impaired fetal lung maturation without

any effect on fetal body weight or litter size 14. This dose is also one tenth of the no

observed adverse effect level (NOAEL) for rodents (50 mg/kg/day), as determined by the

U.S. Environmental Protection Agency (IRIS 2012). Importantly, maternal

concentrations of BPA in our mouse model were determined to be 1.7 ng/ml measured

using gas chromatography–mass spectrometry (GC–MS) 14, which is at the lower end of

the range of those reported in the serum of pregnant women in the US 15.

As a first step in investigating the effects of prenatal BPA exposure on adrenal gland

development and function, we sought to determine changes in adrenal gland weight. We

found that the weight of adrenal glands was significantly increased in both male and

female mice prenatally exposed to BPA when compared to offspring of control mice.

Since BPA did not alter body weight, we observed a similar increase in the ratio of

adrenal gland weight to body weight in prenatally BPA-exposed offspring in both sexes.

This is in marked contrast to the findings of a previous study, which showed that

maternal BPA exposure during pregnancy and lactation led to increases in both adrenal

gland weight and the ratio of adrenal to body weight only in female but not male juvenile

rats 8. Although the precise reasons for the discrepancy between the two studies are not

clear, it is possible that differences in the dosage of BPA and the length of its exposure as

well as the animal species and the offspring age (at which the study was conducted) are

88

important contributing factors. Increased adrenal weight can result in increased adrenal

steroid output, which can be a result of increased output per adrenal cell and/or an

increase in the number of adrenal cells.

We then determined the functional significance of the BPA-induced increases in adrenal

gland weight by examining changes in plasma levels of corticosterone, the principal

glucocorticoid synthesized by the adrenal gland in rodents. We found that although

prenatal exposure to BPA resulted in a significant increase in basal corticosterone levels

in both male and female mice, this increase was greater in BPA exposed males compared

to BPA females. This suggests that plasma corticosterone levels are more vulnerable to

BPA exposure in utero in male than in female offspring. Previous studies reported sex-

dependent changes in plasma corticosterone resulting from developmental exposure to

BPA. For example, one study showed that prandial administration of 40 µg BPA/kg body

weight per day throughout pregnancy and lactation led to elevated plasma corticosterone

levels in juvenile female and but not male rats 8,9. In another study, Zhou, et al. 10 also

observed an increase in basal corticosterone in female juvenile rats exposed to 40 µg

BPA/kg body weight per day throughout pregnancy and lactation; however male rats

were not examined in that study. In contrast, Chen, et al. 11 reported an increase in

corticosterone levels in adult male but not female rats as a result of daily subcutaneous

administration of 2 µg BPA/kg body weight from gestation day 10 to lactation day 7.

These discrepancies can be attributed to differences in the study design, including the

dosage, the timing, the duration, and the mode of BPA administration as well as the age

at which corticosterone levels were measured. Consistent with previous studies, there

were no differences in basal corticosterone levels between control male and female adult

mice 16-18. It is interesting to note that basal corticosterone levels were slightly higher in

both males and females in our study when compared to those published previously 19-21,

which may be attributed to differences in the time of the day when blood samples were

collected, because corticosterone is known to be released in a circadian fashion 22,23.

Furthermore, the presence of varying degrees of potential stressors in the animal housing

environment, such as noise, human traffic and lighting conditions, may also be a

contributing factor 24-26.

89

Given that elevated circulating corticosterone levels are commonly associated with

enhanced ACTH release from the anterior pituitary 27, we measured plasma ACTH levels

and found that they were not altered in either female or male offspring following prenatal

BPA exposure. This suggested that the BPA-induced increases in basal plasma

corticosterone levels are independent of pituitary ACTH, and likely the result of a direct

effect of BPA on the adrenal gland. Our present findings are in marked contrast with

those reported previously showing a concomitant increase in plasma levels of

corticosterone and ACTH in adult male rats 11 and juvenile female rats following

perinatal exposure to BPA 10. As discussed above, differences in the timing, dosage and

duration of BPA exposure likely accounted for the contrasting findings between these

studies.

Our conclusion of BPA exerting a direct effect on the adrenal gland is supported by

previously published in vitro evidence showing that BPA inhibited cortisol and

corticosterone secretion in human adenocarcinoma H295R cells, by inhibiting cyp17A1

(17,20 lyase) 28. Furthermore, BPA reduced the mRNA levels of StAR and cyp11A1, the

two rate-limiting factors in the de novo steroidogenesis, in cultured mouse ovarian

follicles 29,30, while another study reported increased mRNA levels in rat ovarian theca-

interstitial (T-I) cells and granulosa cells following exposure to BPA 31. Furthermore,

BPA has been shown to alter mRNA levels of other steroidogenic P450 enzymes, such as

3β-HSD and 17β-HSD in rat testis 32. In addition, BPA inhibited the activities of 3β-

HSD, CYP17A1 and 17β-HSD3 in both human and rat testis microsomes 33. However, to

date, changes in the expression of StAR and cyp11A1, or any other proteins/enzymes

involved in steroidogenesis, in the adrenal gland following BPA exposure in vivo have

not been examined.

As a first step in examining the effects of prenatal exposure to BPA on steroidogenesis in

the adrenal gland, we sought changes in substrate availability by determining and

comparing levels of perilipin protein between control and BPA exposed offspring.

Perilipin is a protective coating protein surrounding the periphery of lipid droplets, which

are stored in the adrenal gland and are associated with cholesterol ester droplets 13,34. We

found that perilipin protein content was not different between control and BPA exposed

90

mice in either sex, suggesting that cholesterol content, and by inference the substrate

availability for steroidogenesis, is not altered in adult mouse offspring prenatally exposed

to BPA. This is similar to the findings of a previous in vitro study, which showed that

BPA had no effect on perilipin levels in human hepatocyte cells 35. To the best of our

knowledge, this is the first study to examine changes in perilipin expression following

BPA exposure in vivo.

We then examined changes in StAR and cyp11A1, the two rate-limiting steps in

steroidogenesis. We found that adrenal protein levels of both StAR and cyp11A1 were

elevated in female but not male mice, suggesting that the BPA-induced increases in

corticosterone levels in our female offspring are likely the result of an enhanced adrenal

steroidogenesis. Importantly, these findings demonstrate that although prenatal exposure

to BPA alters basal plasma corticosterone levels in both male and female offspring, its

effects on adrenal steroidogenesis are sex-specific. A similar sex-dependent effect was

reported by Xi, et al. 36, who showed that developmental exposure to BPA led to

decreased expression of StAR and cyp11A1 in the testes, whereas no changes in StAR

and an increase in cyp11A1 were detected in the ovaries. However, the lack of a

corresponding increase in StAR and cyp11A1 in the adrenal gland of the male offspring

begs the question of the reasons behind increased corticosterone levels in these animals.

Potential mechanisms/reasons may include changes in one or more of the steroidogenic

enzymes downstream of cyp11A1. Obviously, future studies will be required to address

this issue.

Given that steroidogenic factor-1 (SF-1) is a key transcription factor responsible for the

induction of StAR and cyp11A1 as well as other steroidogenic enzymes 37,38, we

determined if changes in the expression of this transcription factor are responsible for our

observed increases in levels of StAR and cyp11A1 proteins in the adrenal gland of BPA-

exposed female offspring. We found that adrenal levels of SF-1 protein were similar

between control and BPA-exposed mice in both males and females. This suggested that

other transcription factors, such as C/EBPs, Sp1, and DAX1 39,40, may be involved in up-

regulating StAR and cyp11A1 in the adrenal gland of our BPA-exposed female offspring.

Alternatively, BPA-induced phosphorylation of SF-1 could account for the increased

91

level of StAR and cyp11A1 protein, since phosphorylation of SF-1 at the StAR promoter

is required to increase expression of StAR 41,42. Obviously, future studies will be required

to examine these possibilities. It is interesting to note that BPA exposure resulted in

decreased expression of SF-1 in cultured human granulosa cells 43.

2.5 Conclusion In conclusion, the present study demonstrates that prenatal exposure to BPA disrupts

corticosterone homeostasis in the circulation without altering plasma ACTH levels in

both male and female adult mouse offspring. We also provide evidence that BPA disrupts

steroidogenesis independent of SF-1 in a sex-specific manner. Thus, our present findings

provide novel insight into the dynamic effects of developmental exposure to BPA on the

pituitary-adrenal axis development and function.

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25 Laber, K., Veatch, L. M., Lopez, M. F., Mulligan, J. K. & Lathers, D. M. R. Effects of Housing Density on Weight Gain, Immune Function, Behavior, and Plasma Corticosterone Concentrations in BALB/c and C57BL/6 Mice. Journal of the American Association for Laboratory Animal Science : JAALAS 47, 16-23 (2008).

26 Rasmussen, S., Miller, M. M., Filipski, S. B. & Tolwani, R. J. Cage Change Influences Serum Corticosterone and Anxiety-Like Behaviors in the Mouse. Journal of the American Association for Laboratory Animal Science : JAALAS 50, 479-483 (2011).

27 Miller, W. L. & Auchus, R. J. The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocrine reviews 32, 81-151, doi:10.1210/er.2010-0013 (2011).

28 Zhang, X. et al. Bisphenol A disrupts steroidogenesis in human H295R cells. Toxicological sciences : an official journal of the Society of Toxicology 121, 320-327, doi:10.1093/toxsci/kfr061 (2011).

29 Peretz, J. & Flaws, J. A. Bisphenol A down-regulates rate-limiting Cyp11a1 to acutely inhibit steroidogenesis in cultured mouse antral follicles. Toxicology and applied pharmacology 271, 249-256, doi:10.1016/j.taap.2013.04.028 (2013).

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30 Peretz, J., Gupta, R. K., Singh, J., Hernandez-Ochoa, I. & Flaws, J. A. Bisphenol A impairs follicle growth, inhibits steroidogenesis, and downregulates rate-limiting enzymes in the estradiol biosynthesis pathway. Toxicological sciences : an official journal of the Society of Toxicology 119, 209-217, doi:10.1093/toxsci/kfq319 (2011).

31 Zhou, W., Liu, J., Liao, L., Han, S. & Liu, J. Effect of bisphenol A on steroid hormone production in rat ovarian theca-interstitial and granulosa cells. Molecular and cellular endocrinology 283, 12-18, doi:10.1016/j.mce.2007.10.010 (2008).

32 Qiu, L. L. et al. Decreased androgen receptor expression may contribute to spermatogenesis failure in rats exposed to low concentration of bisphenol A. Toxicology letters 219, 116-124, doi:10.1016/j.toxlet.2013.03.011 (2013).

33 Ye, L., Zhao, B., Hu, G., Chu, Y. & Ge, R. S. Inhibition of human and rat testicular steroidogenic enzyme activities by bisphenol A. Toxicology letters 207, 137-142, doi:10.1016/j.toxlet.2011.09.001 (2011).

34 Kraemer, F. B., Khor, V. K., Shen, W. J. & Azhar, S. Cholesterol ester droplets and steroidogenesis. Molecular and cellular endocrinology 371, 15-19, doi:10.1016/j.mce.2012.10.012 (2013).

35 Peyre, L. et al. Comparative study of bisphenol A and its analogue bisphenol S on human hepatic cells: a focus on their potential involvement in nonalcoholic fatty liver disease. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 70, 9-18, doi:10.1016/j.fct.2014.04.011 (2014).

36 Xi, W. et al. Effect of perinatal and postnatal bisphenol A exposure to the regulatory circuits at the hypothalamus-pituitary-gonadal axis of CD-1 mice. Reproductive toxicology (Elmsford, N.Y.) 31, 409-417, doi:10.1016/j.reprotox.2010.12.002 (2011).

37 Calvo, R. M. et al. Screening for mutations in the steroidogenic acute regulatory protein and steroidogenic factor-1 genes, and in CYP11A and dosage-sensitive sex reversal-adrenal hypoplasia gene on the X chromosome, gene-1 (DAX-1), in hyperandrogenic hirsute women. The Journal of clinical endocrinology and metabolism 86, 1746-1749, doi:10.1210/jcem.86.4.7424 (2001).

38 Stocco, D. M. StAR protein and the regulation of steroid hormone biosynthesis. Annual review of physiology 63, 193-213, doi:10.1146/annurev.physiol.63.1.193 (2001).

39 Boucher, J. G., Boudreau, A. & Atlas, E. Bisphenol A induces differentiation of human preadipocytes in the absence of glucocorticoid and is inhibited by an estrogen-receptor antagonist. Nutrition & diabetes 4, e102, doi:10.1038/nutd.2013.43 (2014).

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40 Somm, E. et al. Perinatal exposure to bisphenol a alters early adipogenesis in the rat. Environmental health perspectives 117, 1549-1555, doi:10.1289/ehp.11342 (2009).

41 Gyles, S. L. et al. ERKs regulate cyclic AMP-induced steroid synthesis through transcription of the steroidogenic acute regulatory (StAR) gene. The Journal of biological chemistry 276, 34888-34895, doi:10.1074/jbc.M102063200 (2001).

42 Morohashi, K. I. & Omura, T. Ad4BP/SF-1, a transcription factor essential for the transcription of steroidogenic cytochrome P450 genes and for the establishment of the reproductive function. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 10, 1569-1577 (1996).

43 Kwintkiewicz, J., Nishi, Y., Yanase, T. & Giudice, L. C. Peroxisome proliferator-activated receptor-gamma mediates bisphenol A inhibition of FSH-stimulated IGF-1, aromatase, and estradiol in human granulosa cells. Environmental health perspectives 118, 400-406, doi:10.1289/ehp.0901161 (2010).

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3 BISPHENOL A INDUCES STEROIDOGENIC ACUTE REGULATORY PROTEIN (StAR) EXPRESSION VIA AN UNKNOWN MECHANISM INDEPENDENT OF TRANSCRIPTION, TRANSLATION AND PROTEIN HALF-LIFE IN HUMAN ADRENAL CORTICAL CELLS1

1 The material in this chapter is based on a manuscript submitted to Steroids: Medwid S, Guan H, Yang K. Bisphenol A induces steroidogenic acute regulatory protein (StAR) expression via an unknown mechanism independent of transcription, translation and protein half-life in human adrenal cortical cells (2017).

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3.1 Introduction In Chapter 2, I demonstrated that prenatal BPA exposure increased StAR protein

expression in adult female offspring. Given that StAR is the rate-limiting step in adrenal

steroidogenesis, in the following chapter, I sought to determine the molecular

mechanisms underlying BPA-induced StAR expression using an in vitro cell model

system

Bisphenol A (BPA) is a widespread endocrine disrupting chemical, and a source of major

health concerns. BPA is commonly used in polycarbonate plastics and epoxy resins, such

as plastic containers and the inner-lining of food cans 1-5. BPA is present in human saliva,

urine, and plasma. More concerning is the presence of BPA in placenta, cord blood,

amniotic fluid and breast milk 6-9, raising serious concerns about exposure to BPA in

utero and during critical periods of postnatal development 10. Indeed, numerous human

epidemiological studies have demonstrated an association between gestational exposure

to BPA and adverse health outcomes including pregnancy complications, male genital

abnormalities, childhood obesity, childhood asthma and altered neurological development

in children and adults 11-13.

We recently showed that prenatal BPA exposure led to altered adrenal gland development

and function in adult mouse offspring 14. Specifically, plasma levels of corticosterone

were elevated concomitant with increased adrenal levels of steroidogenic acute regulatory

protein (StAR), the rate-limiting step in steroidogenesis, in adult female offspring 14.

StAR is responsible for the transport of free cholesterol from the outer mitochondrial

membrane (OMM) to the inner mitochondria membrane (IMM), the first and the rate-

limiting step in steroidogenesis 15. BPA is known to alter StAR mRNA and protein levels

in various reproductive tissues, and these effects appear to be species-, sex-, dose- and

exposure time-specific 16-27. However, the molecular mechanisms underlying the effects

of BPA on steroidogenesis, and particularly StAR expression are largely unknown.

Therefore, the present study was designed to address this important question using a

human adrenal cortical cell line as an in vitro model system.

98

3.2 Methods

3.2.1 Reagents Bisphenol A was purchased from Sigma-Aldrich Canada Ltd. (>99% purity; CAS 80-05-

7; Oakville, ON) and dissolved in ethanol to prepare a 10mM stock solution, and stored

at -20°C. ICI 182, 760 (ICI) was purchased from Tocris (cat. no. 1047) and dissolved in

DMSO to prepare a 100mM stock, and stored at -20°C. 4,4',4''-(4-Propyl-[1H]-pyrazole-

1,3,5-triyl)trisphenol) (PPT) and 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN) were

purchased from Tocris (cat. no. 1426 and 1494, respectively) and dissolved in ethanol to

a concentration of 5 mM and 100 mM, respectively, and stored at -20°C. Cycloheximide

(CHX) was purchased from Sigma (C-0934) and dissolved in ethanol to prepare a 100

mM stock and stored at -20°C.

3.2.2 Cell Culture The adrenocortical human cell line NCI-H295 cell line was derived from an adrenal

tumor of a 48-year-old female and was first described by Gazdar et al. 28. The NCI-H295

cell line expresses all steroidogenic enzymes present in the human fetal adrenal glands

and is an established model to study adrenal steroidogenesis 29. The subline, NCI-H295A,

was further derived and characterized from the H295R cell line, and is currently the best

available model of human fetal adrenal gland cells 30. H295A cells (generously provided

by Dr. Walter L. Miller) were cultured in RPMI 1640 media (Invitrogen) with 2% fetal

bovine serum (FBS; Sigma), 0.1% insulin-transferrin-selenium supplement (Sigma

I18884) and 100IU penicillin and 100µg/mL streptomycin (Invitrogen) at 37°C (5%

CO2). Growth medium was replaced every other day.

3.2.3 Western Blot Analysis Levels of various proteins were analyzed using standard western blot analysis, as

previously described 31. Briefly, cells were lysed in SDS gel loading buffer (50 mM Tris-

HCL, pH 6.8, 2% wt/vol SDS, 10% vol/vol glycerol, 100 mM DTT and 0.1% wt/vol

bromophenol blue) to be loaded in a standard SDS-PAGE gel. Protein was then

transferred to a PVDF transfer membrane (Amersham Hybond-P, cat. no. RPN303F, GE

Healthcare Lifesciences, Baie D'Urfe, QC), and blocked overnight with 5% milk in TTBS

(0.1% vol/vol Tween-20 in TBS). Membranes were then probed with primary antibodies

99

for 1-2 hours at room temperature (Table 3.1). Washing was done with TTBS, 3×10

minutes before labeling with horseradish peroxidase-labeled secondary antibody (Table

3.1) for 1 hour at room temperature. After 3×10 minute TTBS washes, proteins were

detected using ECL and visualized using chemiluminescence (cat. no. WBLUR0500,

Luminata Crescendo, Western HRP Substrate; Millipore, Etobicoke, ON) and captured

on the VersaDoc Imaging System (BioRad). Densitometry was performed using Image

Lab Software, comparing levels of proteins expressed as percent of controls.

100

Table 3.1: Primary and secondary antibodies used for western blotting.

Antibody Company Catalog Number Dilution Used

StAR Santa Cruz Sc-25806 1:4000

GAPDH Cell Signaling 14C10 1:10000

Cyp11A1 Bioss Bs-3608R 1:200

3β-HSD Santa Cruz Sc-30820 1:500

ERα Santa Cruz Sc-543 1:500

ERβ Santa Cruz Sc-8974 1:1000

SF-1 Abcam Ab168380 1:1000

C/EBPβ Snata Cruz Sc-150 1:500

Sp1 Millipore 17-601 1:8000

AKAP149 Santa Cruz Sc-377450 1:100

β-tubulin Imgenex IMG-5810A 1:1000

Anti-Rabbit R&D systems HAF008 1:3000

Anti-goat Millipore AP180P 1:8000

Anti-mouse BIO RAD 170-6516 1:7500

101

3.2.4 Real time quantitative RT-PCR The relative abundance of various mRNAs was determined by a two-step real time

quantitative RT-PCR (qRT-PCR), as described previously 32, with the following

modifications. Briefly, total RNA was extracted from cells using RNeasy Mini Kit

(Qiagen Inc., Mississauga, ON) coupled with on-column DNase digestion with the

RNase-free DNase Set (Qiagen) according to the manufacturer’s instructions. One

microgram of total RNA was reverse-transcribed in a total volume of 20 µl using the

High Capacity cDNA Archive Kit (Applied Biosystems, Forest City, CA) following the

manufacturer’s instructions. For every RT reaction set, one RNA sample was set up

without reverse-transcriptase enzyme to provide a negative control. Gene transcript levels

of GAPDH (housekeeping gene whose expression level was found to be stable across

all treatment groups), and StAR were quantified separately by pre-designed and validated

TaqMan® Gene Expression Assays (Applied Biosystems; GAPDH Hs02758991_g1;

StAR Hs00986559_g1) following the manufacturer’s instructions. Briefly, gene

expression assays were performed with the TaqMan® Gene Expression Master Mix

(Applied Biosystems P/N #4369016) and the universal thermal cycling condition (2 min

at 50 °C and 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C)

on the ViiA™ 7 Real-Time PCR System (Applied Biosystems).

The relative amount of StAR and GAPDH mRNA was quantified by the comparative

CT method (also known as ΔΔ CT method) using the Applied Biosystems relative

quantitation and analysis software according to the manufacturer’s instructions. The

amount of StAR mRNA was normalized to GAPDH (housekeeping gene) for each

treatment group. StAR mRNA in BPA treatment is expressed relative to the amount of

transcript present in the control.

3.2.5 Statistical Analysis Results are presented as group means ± SEM for each treatment group, as indicated.

Control and BPA groups were compared using a Student’s t-test or a one-way ANOVA,

followed by a Tukey’s post hoc; statistical significance was set at P<0.05. Statistical

analysis was performed using statistical software PRISM 1992-2008 GraphPAD

Software.

102

3.3 Results

3.3.1 Concentration-dependent effects of BPA on StAR protein expression

To validate this in vitro model system, we determined the effects of various

concentrations of BPA on StAR protein levels in H295A cells. We found that treatment

with increasing concentrations of BPA (1-1000 nM) for 48 h resulted in a concentration-

dependent increase in StAR protein levels (Figure 3.1).

103

Figure 3.1: Concentration-dependent effects of BPA on StAR protein expression.

H295A cells were treated with various concentrations of BPA (1 – 10000 nM) for 48 h.

At the end of treatment, levels of StAR protein were determined by western blotting.

Data are presented as means ± SEM (**P<0.01, ***P<0.001; n=4 independent

experiments).

0 1 10 100 1000 100000

50

100

150

200

250

*****

BPA (nM)

******

StAR

/GAP

DH

(%

cont

rol)

StAR

GAPDH

104

3.3.2 Effects of BPA on selected key steroidogenic enzymes To assess the effects of BPA on adrenal steroidogenesis, protein levels of the two key

steroidogenic enzymes, Cyp11A1 and 3β-HSD, were measured following treatment with

10 nM BPA. We showed that protein levels of both Cyp11A1 and 3β-HSD were not

different between cells treated with and without BPA (Figure 3.2A & B).

105

Figure 3.2: Effects of BPA on selected key steroidogenic enzymes.

H295A cells were treated with 10 nM of BPA for 48 h. At the end of treatment, protein

levels of Cyp11A1 (A) and 3b-HSD (B) were measured by western blotting. Data are

presented as means ± SEM (n=4 independent experiments).

Control BPA0

50

100

150

3β-H

SD/G

APD

H (%

of c

ontro

l)

Control BPA0

50

100

150

Cyp

11A1

/GAP

DH

(% o

f con

trol)

Cyp11A1 GAPDH GAPDH

3β-HSD A B

106

3.3.3 Effects of BPA on ERα and β protein expression To determine the effects of BPA on ER expression, protein levels of ERα and ERβ were

assessed following BPA treatment. We found that neither ERα (Figure 3.3A) nor ERβ

(Figure 3.3B) protein levels were altered by BPA.

107

Figure 3.3: Effects of BPA on ERα and β protein expression.

H295A cells were treated with 10 nM of BPA for 48 h. At the end of treatment, protein

levels of ERa (A), and ERb (B) were measured by western blotting. Data are presented

as means ± SEM (n=4 independent experiments).

Control BPA0

50

100

150

ERα

/GAP

DH

(%

of c

ontro

l)Control BPA

0

50

100

150

ERβ/

GAP

DH

(% o

f con

trol)

GAPDH ERα

GAPDH ERβ A B

108

3.3.4 Effects of ER agonists and antagonist on StAR protein expression

BPA has been shown to activate both ERα and ERβ 33-35. As a first step in determining if

ER is involved in mediating the effects of BPA on StAR expression, we assessed changes

in StAR expression following treatment with the ERα selective agonist PPT and the ERβ

selective agonist DPN, respectively (Figure 3.4A). We found that both PPT and DPN

increased StAR protein levels. We then examined the effects of the ER antagonist ICI

182, 780 (ICI) on BPA-induced expression of StAR. We showed that ICI completely

prevented BPA-induced increases in levels of StAR protein (Figure 3.4B).

109

Figure 3.4: Effects of ER agonists and antagonist on StAR protein expression.

H295A cells were treated with 10 nM PPT, 10 nM DPN, 10 nM BPA, 100 nM ICI

182,780 (ICI), or BPA plus ICI for 48 h. At the end of treatment, levels of StAR protein

were measured by western blotting (A&B). Data are presented as means ± SEM

(*P<0.05, **P<0.01; n=5 independent experiments).

Control ICI BPAICI+BPA0

50

100

150

200**

StAR

/GAP

DH

(%co

ntro

l)

Control BPA PPT DPN0

50

100

150

200

* * *

StAR

/GAP

DH

(%co

ntro

l)B

A

StAR GAPDH

StAR GAPDH

110

3.3.5 Effects of BPA on key StAR transcription factors and StAR mRNA levels

To explore the molecular mechanisms underlying BPA-induced StAR protein expression,

we determined the effects of BPA on the expression of key StAR transcription factors 15,36 and StAR mRNA. Although BPA did not alter SF-1 protein levels (Figure 3.5A), it

increased protein levels of C/EBPβ (Figure 3.5B) and Sp1 (Figure 3.5C). Interestingly,

BPA had no effect on StAR mRNA (Figure 3.5D).

111

Figure 3.5: Effects of BPA on key StAR transcription factors and StAR mRNA

levels.

H295A cells were treated with 10 nM of BPA for 48 h. At the end of treatment, protein

levels of SF-1 (A), C/EBPβ (B) and Sp-1 (C) as well as levels of StAR mRNA (D) levels

were measured by western blotting and qRT-PCR, respectively. Data are presented as

means ± SEM (**P<0.01; n=4 independent experiments).

Control BPA0

50

100

150

SF-1/GAPDH

(%control)

Control BPA 0

50

100

150

StA

R m

RN

A(%

cont

rol)

Control BPA0

50

100

150

200 **C

/EBP

β/GAPDH

(%

cont

rol)

Control BPA0

50

100

150

200 **

Sp-1

/GAP

DH

(%co

ntro

l)

D

SF-1 GAPDH

A

C/EBPβ GAPDH

B

C Sp-1 GAPDH

112

3.3.6 Effects of BPA on key StAR translation protein and StAR pre-protein

We then investigated the possibility that BPA may influence the translation of StAR

protein by examining changes in the key kinase involved in recruiting StAR mRNA and

translating it, namely A-Kinase anchoring protein (AKAP) 149 37,38 as well as the 37-kDa

StAR pre-protein 39. Treatment with BPA did not alter levels of either AKAP149 protein

(Figure 3.6A) or 37-kDa StAR pre-protein (Figure 3.6B).

113

Figure 3.6: Effects of BPA on key StAR translation protein and StAR pre-protein.

H295A cells were treated with 10 nM of BPA for 48 h. At the end of treatment, levels of

AKAP149 protein (A) and 37-kDa StAR pre-protein (B) were measured by western

blotting. Data are presented as means ± SEM (n=4 independent experiments).

Control BPA0

50

100

150

AK

AP

149/β

-Tub

ulin

(% o

f con

trol)

Control BPA0

50

100

150

StAR

/GAP

DH

(% o

f con

trol)

37-kDa StAR GAPDH

B AKAP149 β -Tubulin

A

114

3.3.7 Effects of BPA on half-life of StAR protein

To determine if BPA alters the half-life of StAR protein, cells were treated with BPA for

48 h, followed by co-treatment with the translation inhibitor cycloheximide (CHX) for 2

and 4 hours prior to harvesting for StAR protein measurement. We found that BPA did

not affect the half-life of StAR protein (Control=3.1 h; BPA=3.0 h; Figure 3.7).

115

Figure 3.7: Effects of BPA on half-life of StAR protein.

H295A cells were treated with 10 nM of BPA for 48 h, and then 10 µM cycloheximide

(CHX) was added for 2 h and 4 h prior to harvest. StAR protein levels were measured by

western blotting, and its half-life determined by standard method. Data are presented as

means ± SEM (n=4 independent experiments).

116

3.4 Discussion Adrenal steroidogenesis and most notably glucocorticoid production is critically

controlled by the rate-limiting protein StAR 15,40. We recently demonstrated that prenatal

exposure to BPA resulted in altered adrenal development and function leading to

increased plasma corticosterone levels in adult mouse offspring 14. Furthermore, the

increased plasma corticosterone levels were concomitant with elevated adrenal levels of

StAR protein in female but not male adult offspring 14. This gender-specific changes in

StAR expression is intriguing and begs the question of the molecular mechanisms that

underlie BPA-induced adrenal StAR expression in female offspring. Thus, the present

study was designed to address this important question using the H295A cell line, which is

currently the best available in vitro model of human fetal adrenal cortical cells. Using this

model, we have provided evidence indicating that BPA induces StAR protein expression

via an ER-mediated novel molecular mechanism that does not appear to involve StAR

gene transcription, translation or protein half-life.

The concentration range of BPA (1-10000 nM) used in the present study was consistent

with that used previously in vitro 41, and was lower than what was previously shown to

alter the steroidogenic pathway in non-adrenal cells 16-19. Importantly, the 10 nM BPA

concentration (equivalent to 2.28 ng/ml) used throughout the present study is well within

the range previously reported in urine (0.16-43.42 ng/ml) 42 and plasma (0.5-22.3 ng/ml) 43 of pregnant women.

Since we previously demonstrated that prenatal BPA exposure led to increased StAR

protein levels in adrenal glands of female offspring 14, we sought first to validate our in

vitro model system. Indeed, we found that BPA increased StAR protein levels in a

concentration-dependent manner in H295A adrenal cortical cells. Previously, numerous

studies have examined the effects of BPA treatment on StAR protein levels in

reproductive organs with varying effects 22-27. In vivo studies demonstrated decreased

StAR protein levels in the testes and ovaries of rodents after acute BPA exposure 22-25,27.

In contrast, Qiu et al. 26 observed increased StAR protein levels in the testes of male rats

after chronic BPA treatment. Thus, the effects of BPA exposure on StAR protein appear

to be organ-, tissue- and dose-specific. However, to date no studies have been reported to

117

examine the molecular mechanisms by which BPA induced StAR expression in any

steroidogenic tissues or cells.

BPA has been shown to affect other key steroidogenic enzymes involved in adrenal

glucocorticoid production, including cyp11A1 and 3β-HSD 14,44,45. We also examined

changes in these key enzymes, and found that protein levels of both cyp11A1 and 3β-

HSD were not altered by BPA treatment. It is interesting to note that we previously

reported that prenatal BPA exposure led to increased adrenal levels of cyp11A1 protein

in female but not male adult mouse offspring 14. Furthermore, Lan et al. 45 used the

adrenal mouse cell line, Y1, as well as male mice treated with acute doses of BPA, and

demonstrated an increase in adrenal cyp11A1 protein levels. However, in H295R adrenal

cortical cells, only very high concentrations of BPA (10 µM) were shown to decrease

mRNA levels of cyp11A1 and 3β-HSD 44. Thus, these discrepancies in BPA-induced

changes in cyp11A1 and 3β-HSD may be explained by differences in cell lines, treatment

regime, BPA doses/concentrations and exposure times. Taken together, these findings

suggest that BPA affects adrenal steroidogenesis primarily through altered StAR protein

expression.

BPA is a known agonist for ERβ, and has dual agonistic and antagonistic actions for

ERα, with a higher binding affinity to ERβ than to ERα 34,35. Additionally, human H295R

adrenal cortical cells have been shown to express both ERα and ERβ, with the latter

being the dominant receptor present 46. Therefore, we first examined the effects of BPA

on ERα and ERβ protein expression. We found that levels of both ERα and ERβ protein

were not changed after 48 hours of BPA treatment. ERα null mice showed increased

StAR levels in fetal and adult testes 47,48. Additionally, male transgenic mice expressing

human aromatase, resulting in higher than normal levels of estrogen, had decreased StAR

levels 47,48.However, whether a similar phenotype is present in the adrenal glands is

unknown. We then examined the effects of ERα- and ERβ-specific agonists, PPT and

DPN, on StAR protein. We found that both PPT and DPN increased StAR protein levels,

indicating that both ERα and ERb are involved in regulating StAR expression. To

determine if BPA signals through ER to increase StAR expression, cells were treated

with BPA in the presence and absence of the ER antagonist ICI. We found that ICI

118

abrogated the stimulatory effects of BPA on StAR protein levels. Taken together, these

results suggest that BPA induces StAR protein expression via ERα and/or ERb in adrenal

cortical cells.

StAR is thought to be largely regulated transcriptionally, with numerous transcription

factors binding to the StAR promoter 15,37,40,49. SF-1 is a master regulator of adrenal

steroidogenesis, since SF-1 null mice exhibited diminished basal levels of StAR 15,37,40,50.

Other key transcription factors involved in StAR regulation include C/EBPβ and Sp1 15,37,40. Therefore, we investigated the effects of BPA on these three key transcription

factors. We found that protein levels of SF-1 were unchanged by BPA, which is

consistent with what we and others have previously demonstrated in experimental animal

models 14,51. In contrast, we found protein levels of C/EBPβ and Sp1 were increased after

48 hours of BPA treatment. Increased levels of C/EBPβ were also observed in 3T3-L1

pre-adipocyte cells after BPA treatment 52. C/EBPβ expression was decreased in livers of

male, but not female, offspring after prenatal BPA exposure 53. Previously, Sp1 was

shown to be decreased in resorbed embryos but unchanged in viable embryos 54. Taken

together, these findings suggest that BPA alters the expression of specific transcription

factors known to regulate StAR transcription.

As a first step in determining if BPA affects StAR transcription, we measured StAR

mRNA levels. StAR mRNA was found to be unchanged after 48 hours of BPA treatment.

This is consistent with a previous study in H295R adrenal cortical cells, which showed

that low concentrations of BPA (0.1-1 µM) did not affect StAR mRNA levels, while

higher concentrations of BPA (10 µM) decreased StAR mRNA 44. Other studies in

reproductive tissues also demonstrated no significant changes in StAR mRNA after BPA

treatment 20,21; however, StAR protein levels were not measured in those studies. In

contrast, numerous studies in reproductive cells showed altered StAR mRNA levels, but

results varied greatly in direction and magnitude of change 16-19. Interestingly, StAR

protein levels have been shown to be altered, independent of changes in StAR mRNA,

after exposure to oxysterols 55, pesticides 56, endotoxins 57 and prostaglandins 58;

however, the mechanisms behind these effects were not examined. Collectively, these

findings suggest that BPA regulate StAR primarily at post-transcriptional levels.

119

Recent evidence has pointed to a potential post-transcriptional regulation of StAR 37,40.

One potential player is AKAP149, a mitochondrial anchoring protein, which recruits

StAR mRNA to the OMM 37,38. AKAP149 at the OMM promotes the translation of StAR

by binding the 3’ untranslated region of StAR mRNA to the K homology (KH) RNA-

binding motif 37,38,59. Therefore, we investigated the effects of BPA on AKAP149 protein

levels, which we found to be unaltered after 48 hours of BPA treatment. However, we

recognized that the activity of AKAP121/149 as well as protein levels and the activity of

other key translational regulators were not examined in this study. StAR mRNA is

translated into a 37-kDa pre-protein at the OMM 37,60. Thus, to ascertain the effects of

BPA on StAR translation, we determined the level of the 37-kDa StAR pre-protein. We

did not observe any changes in the 37-kDa StAR pre-protein levels after treatment for 48

hours with BPA. Taken together, these results suggest that BPA does not affect the

translation of StAR protein in adrenal cortical cells.

To further investigate the molecular mechanisms underlying BPA-induced StAR protein

expression, we determined the effects of BPA on StAR protein degradation. The

degradation of the mature StAR protein occurs in the mitochondrial matrix by the two

proteases, LON and AG132, as well as one or more unknown mitochondrial proteases 61,62. As an indicator of protein degradation, the half-life of 30-kDa StAR protein was

determined following treatment with BPA. No differences in the half-life of StAR protein

were observed between control and BPA-treated cells. Taken together, these results

indicate that BPA does not alter the degradation of StAR protein in H295A cells.

Collectively, the present findings suggest that BPA does not regulate StAR protein levels

through changes in transcription, translation or degradation. However, the regulation of

StAR is extremely complex and is not yet fully understood 37,49,63. Thus, BPA may be

acting through an unknown mechanism to modulate StAR expression, and obviously

future studies will be required to examine this possibility. Additionally, due to the short

half-life of both StAR pre- and mature protein 64, transcription and translation markers

were only examined at 48 hours, however it is possible that changes in mRNA or 37-kDa

StAR pre-protein could happen at earlier time points. Furthermore, we did not examine

120

the effects of BPA on the cleavage of StAR or its transportation from the OMM to the

IMM. However, since we found no changes in the levels of 37-kDa StAR pre-protein, it

is unlikely that BPA affects either of these two steps. Furthermore, this study did not

measure the 32-kDa isoform of StAR protein, which, if changed, may provide a potential

explanation for BPA-induced changes in mature StAR protein levels 60,64.

In conclusion, the present study provides strong evidence that BPA signals through ERa

and/or ERb to increase StAR protein levels in H295A cells via an unknown mechanism

independent of StAR gene transcription, translation and protein half-life.

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4 Wetherill, Y. B. et al. In vitro molecular mechanisms of bisphenol A action. Reproductive toxicology (Elmsford, N.Y.) 24, 178-198, doi:10.1016/j.reprotox.2007.05.010 (2007).

5 Rubin, B. S. Bisphenol A: an endocrine disruptor with widespread exposure and multiple effects. The Journal of steroid biochemistry and molecular biology 127, 27-34, doi:10.1016/j.jsbmb.2011.05.002 (2011).

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38 Ginsberg, M. D., Feliciello, A., Jones, J. K., Avvedimento, E. V. & Gottesman, M. E. PKA-dependent binding of mRNA to the mitochondrial AKAP121 protein. Journal of molecular biology 327, 885-897 (2003).

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41 Welshons, W. V., Nagel, S. C. & vom Saal, F. S. Large effects from small exposures. III. Endocrine mechanisms mediating effects of bisphenol A at levels of human exposure. Endocrinology 147, S56-69, doi:10.1210/en.2005-1159 (2006).

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43 Padmanabhan, V. et al. Maternal bisphenol-A levels at delivery: a looming problem? Journal of perinatology : official journal of the California Perinatal Association 28, 258-263, doi:10.1038/sj.jp.7211913 (2008).

44 Feng, Y. et al. Effects of bisphenol analogues on steroidogenic gene expression and hormone synthesis in H295R cells. Chemosphere 147, 9-19, doi:10.1016/j.chemosphere.2015.12.081 (2016).

45 Lan, H. C., Lin, I. W., Yang, Z. J. & Lin, J. H. Low-dose Bisphenol A Activates Cyp11a1 Gene Expression and Corticosterone Secretion in Adrenal Gland via the JNK Signaling Pathway. Toxicological sciences : an official journal of the Society of Toxicology 148, 26-34, doi:10.1093/toxsci/kfv162 (2015).

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47 Strauss, L. et al. Increased exposure to estrogens disturbs maturation, steroidogenesis, and cholesterol homeostasis via estrogen receptor alpha in adult mouse Leydig cells. Endocrinology 150, 2865-2872, doi:10.1210/en.2008-1311 (2009).

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51 Durando, M. et al. Neonatal expression of amh, sox9 and sf-1 mRNA in Caiman latirostris and effects of in ovo exposure to endocrine disrupting chemicals. General and comparative endocrinology 191, 31-38, doi:10.1016/j.ygcen.2013.05.013 (2013).

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127

4 BISPHENOL A STIMULATES ADRENAL CELL PROLIFERATION THROUGH ERb-MEDIATED ACTIVATION OF THE SONIC HEDGEHOG SIGNALING PATHWAY1

1 The material in this chapter is based on a manuscript submitted to Journal of Steroid Biochemistry and Molecular Biology: Medwid S, Guan H, Yang K. Bisphenol A stimulates adrenal cortical cell proliferation via ERβ-mediated activation of the sonic hedgehog signaling pathway (2017).

128

4.1 Introduction In Chapter 2, I observed an increase in adrenal gland weight in adult mouse offspring

prenatally exposed to BPA, with no changes in basal plasma ACTH levels. In this

chapter, I sought to determine the cellular and molecular mechanisms that underlie BPA-

induced aberrant adrenal gland developmental phenotype using an in vitro cell model

system.

Bisphenol A (BPA) is one of the most well-known and prevalent endocrine disrupting

chemicals, and has gained universal attention due to its adverse effects in humans and

experimental animal models 1. BPA is widely used in the production of polycarbonate

plastics and epoxy resins, such as food and beverage storage containers and thermal paper

receipts 1,2. Biomonitoring studies have detected BPA in human saliva, milk, serum and

urine collected globally 2. More alarming is the presence of BPA in human fetal blood,

placental tissue and amniotic fluid 2,3. This has raised serious concerns about the impact

of BPA exposure on the developing fetus during the critical period of organ maturation.

Indeed, numerous studies have shown that BPA exerts adverse effects on many fetal

organ systems, including the brain 4,5, lungs 6, liver 7, pancreas 8, heart 9, adrenal gland 10,11, mammary gland 12,13, and ovary 14,15.

We recently showed that prenatal exposure to BPA resulted in altered adrenal gland

structure and function in adult mouse offspring 10. Specifically, absolute and relative

adrenal gland weight was increased in both male and female adult offspring 10. Similarly,

Panagiotidou et al. reported adrenal hyperplasia in juvenile female rat offspring following

exposure to BPA during pregnancy and lactation 11. Alterations in adrenal weight and

structure is normally associated with changes in plasma levels of adrenocorticotrophic

hormone (ACTH). However, we did not observe an increase in basal plasma levels of

ACTH, and concluded that BPA may directly affect adrenal gland weight independent of

plasma ACTH in our prenatally BPA exposed mouse model 10. BPA has previously been

shown to increase cell proliferation in various tissues, including breast cancer 16-18,

ovarian cancer 19,20, neuroblastoma 21, Hela 22, prostate cancer 23, seminoma 24 and sertoli

cells 25. However, the effects of BPA on adrenal cortical cell proliferation has never been

examined.

129

Sonic hedgehog (shh) signaling pathway is a key mediator of embryonic development, as

well as cell maintenance and tissue repair in adults 26,27. Specifically, the shh pathway is

found to be activated during development, as well as in various forms of cancer due to its

role in promoting cell proliferation through direct transcriptional activation of

proliferation factors cyclin D1 and cyclin D2 26. Shh signaling components (shh, Gli,

Patched 1) have been detected in human adrenal cortical cell lines, human fetal and adult

adrenal glands, as well as both pediatric and adult adrenal tumors 28,29. Evidence of shh

involvement in adrenal cell proliferation is demonstrated by the presence of an adrenal

cortex hypotrophy phenotype in shh null mice 30,31. Thus, the present study was

undertaken to determine (1) if BPA promotes adrenal cell proliferation, which may help

explain the increased adrenal gland weight phenotype we reported in our previous study 10; and (2) if so, whether the stimulatory effects of BPA on adrenal cortical cell

proliferation are mediated through ERβ-mediated activation of the shh pathway using a

human adrenal cortical cell line as an in vitro model system.

4.2 Methods

4.2.1 Reagents

Bisphenol A was purchased from Sigma-Aldrich Canada Ltd. (CAS 80-05- 7; Oakville,

ON) and dissolved in ethanol to prepare 10 mM stock solution, and stored at -20°C.

Cyclopamine was purchased from Toronto Research Chemicals (C988400; Toronto,

ON), dissolved in ethanol to prepare 10 mM stock solution and stored at -20°C. 2,3-bis(4-

Hydroxyphenyl)-propionitrile (DPN) and 4-[2-Phenyl-5,7-

bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP) were purchased from

Tocris Bioscience (cat. no. 1494; Minneapolis, MN) and Abcam (ab145148; Toronto,

ON), respectively, dissolved in ethanol to a concentration of 100 mM, and stored at -

20°C.

4.2.2 Cell Culture

The adrenocortical human cell line NCI-H295 cell line was derived from an adrenal

tumor of a 48-year-old female and was first described by Gazdar et al. 32. The NCI-H295

cell line expresses all steroidogenic enzymes present in the human fetal adrenal glands

130

and is an established model to study adrenal steroidogenesis 33. The subline, NCI-H295A,

was further derived and characterized from the H295R cell line, and is currently the best

available model of human fetal adrenal gland cells 34. H295A cells (generously provided

by Dr. Walter L. Miller) were cultured in RPMI 1640 media (Invitrogen) with 2% fetal

bovine serum (FBS; Sigma), 0.1% insulin-transferrin-selenium supplement (Sigma

I18884) and 100IU penicillin and 100µg/mL were starved in serum-free media 24 h

before treatment, and cultured in 0.2% FBS media throughout treatments.

4.2.3 Western Blot Analysis

Levels of various proteins were analyzed using standard western blot analysis, as

previously described 35. Briefly, cells were lysed in SDS gel loading buffer (50mM Tris-

HCL, pH 6.8, 2% wt/vol SDS, 10% vol/vol glycerol, 100mM DTT and 0.1% wt/vol

bromophenol blue) to be loaded to a standard SDS-PAGE gel. Protein was then

transferred to a PVDF transfer membrane (Amersham Hybond-P, cat. no. RPN303F, GE

Healthcare Lifesciences, Baie D'Urfe, QC), and blocked overnight with 5% milk in TTBS

(0.1% vol/vol Tween-20 in TBS). Membranes were then probed with primary antibodies

for 1-2 hours at room temperature (Table 4.1). Washing was done with TTBS, 3×10

minutes before labeling with horseradish peroxidase-labeled secondary antibody (Table

4.1), for 1 hour at room temperature. After 3×10 minute TTBS washes, protein were

detected using ECL and visualized using chemiluminescence (cat. no. WBLUR0500,

Luminata Crescendo, Western HRP Substrate; Millipore, Etobicoke, ON) and captured

on the VersaDoc Imaging System (BioRad). Densitometry was performed using Image

Lab Software, comparing levels of proteins expressed as percent of controls.

131

Table 4.1: Primary and secondary antibodies used for western blotting.

Antibody Company Catalog Number Dilution Used

PCNA Cell Signalling 2586 1:5000

GAPDH Imgenex IMG-5567 1:10000

Cyclin D1 Santa Cruz Sc-717 1:500

Cyclin D2 Cell Signalling 3741 1:1000

ERβ Santa Cruz Sc-8974 1:1000

Gli1 Abcam ab49314 1:500

Shh Santa Cruz Sc-365112 1:200

β-tubulin Imgenex IMG-5810A 1:1000

Lamin B1 Abcam Ab16048 1:20000

Anti-Rabbit R&D systems HAF008 1:3000

Anti-mouse BIO RAD 170-6516 1:7500

132

4.2.4 Cell Number Assessment

Cells were seeded in 2% FBS-RMPI 1640 culture medium and were incubated overnight.

After 24 h serum starvation, the medium was changed to 0.2% FBS RMPI 1640

containing 10 nM BPA. After 72 h incubation, the cells trypsinized, added in equal

volumes to trypan blue stain 0.4% (Invitrogen T10282) and counted with Countess

Automated cell counter (Invitrogen C10277).

4.2.5 Real-time quantitative RT-PCR

The relative abundance of various mRNAs was determined by a two-step real time

quantitative RT-PCR (qRT-PCR), as described previously 36, with the following

modifications. Briefly, total RNA was extracted from cells using RNeasy Mini Kit

(Qiagen Inc., Mississauga, ON) coupled with on-column DNase digestion with the

RNase-free DNase Set (Qiagen) according to the manufacturer’s instructions. One

microgram of total RNA was reverse-transcribed in a total volume of 20 µl using the

High Capacity cDNA Archive Kit (Applied Biosystems, Forest City, CA) following the

manufacturer’s instructions. For every RT reaction set, one RNA sample was set up

without reverse-transcriptase enzyme to provide a negative control. Gene transcript levels

of GAPDH, GLI1 and SHH were quantified separately by pre-designed and validated

TaqMan® Gene Expression Assays (Applied Biosystems; Table 4.2) following the

manufacturer’s instructions. Briefly, gene expression assays were performed with the

TaqMan® Gene Expression Master Mix (Applied Biosystems P/N #4369016) and the

universal thermal cycling condition (2 min at 50 °C and 10 min at 95 °C, followed by 40

cycles of 15 s at 95 °C and 1 min at 60 °C) on the ViiATM 7 Real-Time PCR System

(Applied Biosystems).

The relative amounts of various gene-specific mRNAs in each RNA sample was

quantified by the comparative CT method (also known as ΔΔ CT method) using the

Applied Biosystems relative quantitation and analysis software according to the

manufacturer’s instructions. For each experiment, gene specific mRNAs were normalized

to the housekeeping gene GAPDH. The amount of various gene-specific mRNAs under

different treatment conditions is expressed relative to the amount of transcript present in

the untreated control.

133

Table 4.2: TaqMan® gene expression assays for the human genes analyzed.

Gene Name Assay ID

SHH Hs00179843_m1

GLI1 Hs00171790_m1

GAPDH Hs02758991_g1

134

4.2.6 Statistical Analysis

Results are presented as group means ± SEM of four independent experiments, as

indicated. Data was analyzed using a Student’s t-test or a one-way ANOVA, followed by

a Tukey’s post hoc; statistical significance was set at P<0.05. Statistical analysis was

performed using statistical software GraphPad Prism Version 5 Software.

4.3 Results

4.3.1 Time- and concentration-dependent effects of BPA on cell proliferation.

As a first step in determining the effects of BPA on cell proliferation, protein levels of

PCNA, a universal marker of cell proliferation, were assessed over time. Levels of PCNA

protein were unchanged at 24 and 48 h, but were significantly elevated at 72 h following

treatment with 10 nM of BPA (Figure 4.1A). A similar trend of change was observed in

cell number following BPA treatment (Figure 4.1B). We then treated cells with

increasing concentrations of BPA (1-1000 nM) for 72 h, and showed that this treatment

resulted in a concentration-dependent increase in PCNA protein levels such that the

maximal effect was observed at 10 nM BPA (Figure 4.1C).

135

Figure 4.1: Time- and concentration-dependent effects of BPA on cell proliferation.

H295A cells were treated with 10 nM of BPA for various times (24-72 h) or increasing

concentrations (1-1000 nM) of BPA for 72 h. At the end of treatment, levels of PCNA (a

universal marker of proliferation) (A, C) and cell number (B) were determined by

western blotting and cell counting, respectively. Data are presented as mean ± SEM

(*P<0.05, ***P<0.001 vs. control; n=4 independent experiments).

24 h 48 h 72 h0

50

100

150

200***

ControlBPA

PCN

A/G

APD

H(%

of c

ontro

l)B

A PCNA

GAPDH

24 h 48 h 72 h0

100

200

300

400

500ControlBPA *

Cel

l Num

ber

(% o

f con

trol)

0 1 10 100 10000

60

120

180

BPA concentration (nM)

***

PCN

A/G

APD

H(%

of c

ontro

l)

C PCNA

GAPDH

136

4.3.2 Effects of BPA on the expression of key cell proliferation factors.

To further examine the effects of BPA on cell proliferation, protein levels of the two key

proliferation factors, cyclin D1 and cyclin D2, were determined. Although levels of both

cyclin D1 and cyclin D2 proteins were unchanged after 48 h of BPA treatment (Figure

4.2A), they were significantly increased after 72 h of BPA treatment (Figure 4.2B).

137

Figure 4.2: Effects of BPA on key cell proliferation factors.

H295A cells were treated with 10 nM of BPA for 48 h (A) or 72 h (B). At the end of

treatment, levels of the two key cell proliferation factors, cyclin D1 and cyclin D2 were

determined by western blotting. Data are presented as mean ± SEM (**P<0.01 vs.

control; n=4 independent experiments).

C BPA0

50

100

150

200 **

Cyc

lin D

2/β-

tubu

lin (%

of c

ontro

l)

C BPA0

50

100

150C

yclin

D1/β-

tubu

lin (%

of c

ontro

l)

C BPA0

50

100

150

200

**

Cyc

lin D

1/β-

tubu

lin (%

of c

ontro

l)

Cyclin D2 β-tubulin

Cyclin D1 Cyclin D2 β-tubulin

Cyclin D1

72 h

A B

C BPA0

50

100

150

Cyc

lin D

2/β-

tubu

lin (%

of c

ontro

l)

48 h

138

4.3.3 Effects of BPA on selected components of the shh signaling pathway.

Shh signaling is known to be essential for adrenal development and proliferation. Adrenal

specific shh knockout mice display severe adrenal hypoplasia, specifically an

underdeveloped cortex in fetal and adult mice 30,37. To explore the role of shh signaling in

mediating BPA-induced cell proliferation, changes in key shh signaling pathway

components were examined. Levels of shh mRNA, but not Gli1 mRNA, were increased

at 48 h post BPA treatment (Figure 4.3A&B). In contrast, protein levels of both shh and

Gli1 were elevated following 48 h of BPA treatment (Figure 4.3C&D), which returned

to control levels at 72 h (Figure 4.3E&F).

139

Figure 4.3: Effects of BPA on selected components of the shh signaling pathway.

H295A cells were treated with 10 nM of BPA for 48 h. At the end of treatment, levels of

shh mRNA (A) and Gli1 mRNA (B) were determined by qRT-PCR. Levels of shh

protein (C&E) and Gli1 protein (D&F) at 48 h (C&D) and 72 h (E&F) were determined

by western blotting. Data are presented as mean ± SEM (*P<0.05, **P<0.01, vs. control;

n=4 independent experiments).

C BPA0

50

100

150

200 *

shh

mR

NA

(% o

f con

trol)

C BPA0

50

100

150

Gli1

mR

NA

(% o

f con

trol)

C BPA0

50100150200250

**

Gli1

/β-tu

bulin

(% o

f con

trol)

C BPA0

50

100

150

200*

shh/β-

tubu

lin (%

of c

ontro

l)C D shh

β-tubulin β-tubulin Gli1

A B

C BPA0

50

100

150

shh/β-

tubu

lin (%

of c

ontro

l)

C BPA0

50

100

150

Gli1

/β-tu

bulin

(% o

f con

trol)

shh β-tubulin

E

β-tubulin Gli1

F

140

4.3.4 Effects of BPA on activity of the shh signaling pathway.

Activation of the shh signaling pathway involves translocation of the shh transcription

factor Gli1 from cytoplasm to the nucleus where it acts as an activator of shh target genes 38,39. To determine if BPA activates the shh signaling pathway, we measured Gli1 protein

levels in cytosolic and nuclear fractions following treatment with BPA for 48 h. BPA

treatment significantly increased Gli1 protein in nuclear but not cytosolic fraction

(Figure 4.4A&B). To ascertain if BPA activation of the shh signaling pathway is ligand-

dependent, we used cyclopamine (Cyc), which blocks the shh pathway at the SMO

receptor. We treated cells with BPA in the presence and absence of Cyc, and examined

changes in Gli1 protein. We found that Cyc prevented BPA-induced increases in Gli1

protein levels (Figure 4.4C).

141

Figure 4.4: Effects of BPA on activity of the shh signaling pathway

H295A cells were treated with 10 nM BPA for 48 h. At the end of the treatment, levels of

Gli1 protein in cytosolic (A) and nuclear (B) extracts were determined by western

blotting. Alternatively, H295A cells were treated with either 10 nM BPA, 10 µM

cyclopamine (Cyc) or both for 48 h. At the end of treatment, levels of Gli1 protein were

determined by western blotting (C). Data are presented as mean ± SEM (*P<0.05 vs.

control; different letters indicate statistically significant differences among groups; n=4

independent experiments).

C BPA0

100

200

300*

Gli1

/Lam

in B

1 (%

of c

ontro

l)

C BPA0

50

100

150

Gli1

/GAP

DH

(% o

f con

trol)

A B

GAPDH Lamin B1 Gli1 Gli1

Cytosolic

C Gli1

GAPDH

Nuclear

0

50

100

150

200

C BPACyc Cyc+BPA

aa

ba

Gli1

/GAP

DH

(% o

f con

trol)

142

4.3.5 Effects of shh pathway inhibition on BPA-induced cell proliferation markers.

Shh signaling is known to induce cell proliferation in a variety of tissues 26,40,41.

Specifically, the transcription factor Gli1 is known to directly stimulate the transcription

of cyclin D1 and D2 genes, CCND1 and CCND2 26. To provide functional evidence for

the involvement of the shh signaling pathway in mediating BPA-induced cell

proliferation, we assessed changes in protein levels of PCNA, cyclin D1 and D2

following treatment with BPA in the presence and absence of the shh pathway inhibitor

Cyc. Cyc completely blocked BPA-induced increases in protein levels of PCNA (Figure

4.5A), as well as cyclin D1 and D2 (Figure 4.5B).

143

Figure 4.5: Effects of shh inhibition on BPA-induced cell proliferation markers.

H295A cells were treated with 10 nM BPA, 10 µM cyclopamine (Cyc) or both for 72 h.

At the end of treatment, levels of PCNA (A), cyclin D1 and cyclin D2 (B) were

determined by western blotting. Data are presented as mean ± SEM (Different letters

indicate statistically significant differences among groups; n=4 independent

experiments).

PCNA

GAPDH

Cyclin D1

β-tubulin

Cyclin D2

A

B

0

50

100

150

200

C BPACyc Cyc+BPA

b

a a a

PCN

A/G

APD

H(%

of c

ontro

l)

0

40

80

120

160

C BPACyc Cyc+BPA

b

aa a

Cyc

lin D

1/β-

tubu

lin (%

of c

ontro

l)

0

50

100

150

200

C BPACyc Cyc+BPA

b

a a

Cyc

lin D

2/β-

tubu

lin (%

of c

ontro

l)

ab

144

4.3.6 Effects of BPA on estrogen receptor β expression and activity

The translocation of ER from cytosol to the nucleus is essential for transcriptional

activation of estrogen target genes 42-44. To examine if BPA activates ERβ, we measured

protein levels of ERβ in total cell lysates as well as cytosolic and nuclear fractions

following BPA treatment for 48 h. Although BPA treatment did not alter total ERβ

protein levels (Figure 4.6A), it decreased cytosolic while increasing nuclear levels of

ERβ protein (Figure 4.6B&C).

145

Figure 4.6: Effects of BPA on estrogen receptor β expression and activity.

H295A cells were treated with 10 nM of BPA for 48 h. At the end of treatment, levels of

ERβ protein in total (A) cytosolic (B) and nuclear (C) extracts were subjected to western

blotting. Data are presented as mean ± SEM (**P<.0.01 vs. control; n= 4 independent

experiments).

C BPA0

50100150200250

**

ERβ/

Lam

in B

1 (%

of c

ontro

l)

C BPA0

50

100

150

**

ERβ/

GAP

DH

(% o

f con

trol)

C BPA0

50

100

150

ERβ/

GAP

DH

(% o

f con

trol)

C

A

GAPDH ERβ

GAPDH ERβ

Lamin B1 ERβ

B

Cytosolic

Nuclear

146

4.3.7 Effects of DPN and PHTPP on BPA-induced cell proliferation markers.

We then investigated the involvement of ERβ in BPA-induced cell proliferation using the

ERβ specific agonist DPN and the ERβ specific antagonist PHTPP. Treatment with DPN

significantly increased protein levels of PCNA (Figure 4.7A) as well as cyclin D1 and

cyclin D2 (Figure 4.7C) at 72 h. Furthermore, pretreatment with PHTPP completely

prevented BPA-induced increases in levels of PCNA (Figure 4.7B), cyclin D1 and cyclin

D2 (Figure 4.7D) proteins.

147

Figure 4.7: Effects of DPN and PHTPP on BPA-induced cell proliferation markers.

H295A cells were treated with 10 nM DPN, 10 nM BPA, 100 nM PHTPP, or both

PHTPP and BPA for 72 h. At the end of treatment, levels of PCNA protein (A&B),

cyclin D1 and cyclin D2 protein (C&D) were determined by western blotting. Data are

presented as mean ± SEM (**P<0.01, ***P<0.001 vs. control; different letters indicate

statistically significant differences among groups; n=4 independent experiments).

C DPN0

50

100

150

200***

Cyc

lin D

1/β-

tubu

lin (%

of c

ontro

l)

C DPN0

50

100

150

200 ***PC

NA/

GAP

DH

(% o

f con

trol)

C DPN0

50

100

150

200

**

Cyc

lin D

2/β-

tubu

lin (%

of c

ontro

l)

β-tubulin

A PCNA

GAPDH

C Cyclin D1 Cyclin D2

B PCNA

GAPDH

D Cyclin D1

β-tubulin Cyclin D2

0

50

100

150

200

C BPAPHTPP PHTPP+BPA

b

a a a

Cyc

lin D

2/β-

tubu

lin (%

of c

ontro

l)

0

50

100

150

200

C BPAPHTPP PHTPP+BPA

b

a aC

yclin

D1/β-

tubu

lin (%

of c

ontro

l)ab

050

100150200250

C BPAPHTPP PHTPP+BPA

b

a a

PCN

A/G

APD

H(%

of c

ontro

l)

ab

148

4.3.8 Effects of DPN and PHTPP on BPA-induced activation of the shh signaling pathway.

ERa has been shown to increase shh activity in breast 45,46 and gastric 47 cancer cells.

However, this effect has yet to be shown with ERβ. Therefore, we tested the hypothesis

that BPA acts through ERβ to activate the shh signaling pathway. We showed that the

ERβ specific agonist DPN increased protein levels of both shh and Gli1 after 48 h

treatment (Figure 4.8A&C). Importantly, the ERβ specific antagonist PHTPP completely

blocked BPA-induced increases in both shh and Gli1 protein levels (Figure 4.8B&D).

149

Figure 4.8 Effects of DPN and PHTPP on BPA-induced shh pathway activation.

H295A cells were treated with 10 nM DPN, 10 nM BPA, 100 nM PHTPP, or both

PHTPP and BPA for 48 h. At the end of treatment, levels of shh protein (A&B) and Gli1

protein (C&D) were determined by western blotting. Data are presented as mean ± SEM

(**P<0.01 vs. control; different letters indicate statistically significant differences among

groups; n=4 independent experiments).

C DPN0

50

100

150

200**

shh/β-

tubu

lin (%

of c

ontro

l)

C DPN0

50

100

150 **

Gli1

/GAP

DH

(% o

f con

trol)

Gli1

B A

C

shh β-tubulin

Gli1

GAPDH

D

β-Tubulin shh

GAPDH

050

100150200250

C BPAPHTPP PHTPP+BPA

b

aa a

Gli1

/GAP

DH

(% o

f con

trol)

0

50

100

150

200

C BPAPHTPP PHTPP+BPA

b

a aab

shh/β-

tubu

lin (%

of c

ontro

l)

150

4.4 Discussions

Proper adrenal gland development is essential for adrenal steroidogenesis, particularly

glucocorticoid production in later-life. We recently demonstrated that prenatal exposure

to BPA resulted in abnormal adrenal gland development and function in adult mouse

offspring, including increased adrenal gland weight independent of plasma ACTH levels 10. However, the molecular mechanisms underlying the BPA-induced increase in adrenal

gland weight remain unknown. Therefore, the present study was designed to address this

important question using the best available model of fetal adrenal cortical cells, the

H295A cell line. We have demonstrated that BPA stimulates adrenal cell proliferation via

ERβ-mediated activation of the shh signaling pathway. Thus, our present findings reveal

a plausible molecular mechanism by which BPA influences adrenal gland development

and function.

The concentration of BPA used in this study (10 nM) is in line with those used in

previous in vitro studies 48. Importantly, this concentration (equivalent to 2.28 ng/ml) is

well within the range previously reported in plasma (0.5-22.3 ng/ml) 49 and urine (0.16-

43.42 ng/ml) 50 of pregnant women in North American.

BPA has been shown to influence cell proliferation in both in vivo and in vitro models. In

experimental animal models, prenatal exposure to BPA led to increased cell proliferation

in fetal liver 7, prostate 51, pancreas 52, and pituitary gland 53. In contrast, offspring of rats

exposed to BPA during pregnancy and lactation showed decreased proliferation in neural

stem cells of the hypothalamus and sub-ventricular zone 54. In several in vitro models,

BPA increases cell proliferation at various concentrations 16-25. Interestingly, in sertoli

cells, nanomolar concentrations of BPA induced cell proliferation, while micromolar

concentrations decreased cell proliferation, suggesting that the effect of BPA on cell

proliferation is concentration-dependent 55. To the best of our knowledge, we are the first

to demonstrate that BPA, at environmentally relevant concentrations, significantly

increases cell number as well as the expression of PCNA, cyclin D1 and D2, three key

markers of cell proliferation, in adrenal cortical cells. This indicates that BPA stimulates

adrenal cortical cell proliferation. Thus, our present study provides a plausible cellular

151

mechanism by which prenatal BPA exposure results in increased adrenal gland weight in

adult mouse offspring 10.

Activation of the shh signaling pathway is known to increase the transcription of genes

encoding both cyclin D1 and D2 genes, leading to increased cell proliferation 26.

Recently, BPA has been shown to increase levels of microRNA-107 (miRNA-107),

which inhibits the expression of suppressor of fused homolog (SUFU) and GLI family

zinc finger 3 (Gli3) in human endometrial cancer in RL95-2 cells 56. Both SUFU and Gli3

are repressors of the shh signaling pathway, thus BPA-induced suppression of these

proteins may potentially lead to the activation of shh signaling and consequently

increased proliferation in endometrial cells 56. Therefore, we investigated the possibility

that the BPA-induced adrenal cortical cell proliferation may be mediated via activation of

the shh signaling pathway. As a first step in examining this possibility, we determined the

effects of BPA on shh expression, and found that levels of both shh mRNA and protein

were increased after 48 hours of BPA treatment, which preceded the increase in cell

proliferation we observed at 72 hours.

An increase in shh protein and secretion results in its binding to the transmembrane

receptor Patched 1 (Ptch1), which prevents Ptch1 from inhibiting another transmembrane

protein smoothened (SMO) 38,39. SMO can then be released from the plasma membrane

into the cytoplasm, leading to the release of a complex containing the transcription

factors Gli1-3, allowing them to translocate to the nucleus to regulate transcription of

target genes 38,39. Specifically, the nuclear translocation of the positive transcriptional

regulator Gli1, is considered a marker of shh signaling activation 38,39. Therefore, we

investigated the potential for BPA to alter Gli1 protein and mRNA levels. We found that

although BPA did not alter Gli1 mRNA, it increased Gli1 protein levels at 48 hours. The

regulation of Gli1 at post-transcriptional level is well established and could be a result of

changes in translation and phosphorylation efficiency 57,58. Furthermore, BPA

significantly increased Gli1 protein levels in the nuclear fraction without altering those in

the cytosolic fraction, suggesting that BPA enhanced nuclear translocation of Gli1, and

consequently the activity of the shh signaling pathway. Given the observed increase in

Gli1 protein levels in total cell lysates, the relatively minor and non-significant decrease

seen in cytosolic Gli1 levels is consistent with our notion of an enhanced Gli1 nuclear

152

translocation following BPA treatment. It is known that activation of the shh signaling

pathway is mediated through either the ligand-dependent or the ligand-independent

pathway 59. To determine if BPA acts through the ligand-dependent shh signaling

pathway, we examined the effects of BPA on Gli1 protein levels in the presence and

absence of cyclopamine. Cyclopamine is a potent inhibitor of the shh signaling pathway

by preventing release and translocation of the SMO receptor. In the present study, we

showed that cyclopamine blocked the effects of BPA on Gli1 protein levels, indicating

that BPA activates the shh pathway through the ligand-dependent pathway.

To ascertain whether BPA-induced activation of the shh signaling pathway leads to

increased cell proliferation, we treated cells with BPA in the presence and absence of

cyclopamine. We found that cyclopamine completely abrogated the stimulatory effects of

BPA on cell proliferation, as indicated in protein levels of PCNA, cyclin D1 and D2.

Taken together, these results demonstrate the involvement of the shh signaling pathway

in BPA-induced adrenal cortical cell proliferation.

It is well known that BPA acts as an ERβ agonist, with a higher affinity for ERβ than

ERa 60,61. Furthermore, ERβ is the dominant estrogen receptor expressed in human

H295R adrenal cortical cells 62. Therefore, we then investigated the role of ERβ in BPA-

induced cell proliferation and shh activation. Given that a key step in ERβ activation is its

rapid nuclear translocation upon binding to its ligand 63, we determined the effects of

BPA on ERβ translocation at 48 h. This time point was chosen based on the BPA-

induced increase in shh mRNA at 48 h. We found that levels of ERβ protein were

increased in the nuclear fraction but decreased in the cytosolic fraction following BPA

treatment, indicating that BPA enhanced translocation of ERβ to the nucleus in H295A

cells. However, it is likely that the BPA-induced increase in ERβ nuclear translocation

may have occurred earlier than 48 h.

Although estrogen has previously been shown to increase adrenal cell proliferation in

both animal models 64 and the H295R cell line 62. We then sought to determine if

activation of ERβ stimulates adrenal cell proliferation using the ERβ selective agonist

DPN. We showed that DPN increased protein levels of the three key proliferation

markers, PCNA, cyclin D1 and D2, indicating that the activation of ERβ by DPN led to

153

increased cell proliferation. To provide evidence for the involvement of ERβ in mediating

BPA-induced cell proliferation, we treated cells with BPA in the presence and absence of

the ERβ-specific antagonist PHTPP. We found that PHTPP completely blocked the

stimulatory effects of BPA on PCNA, cyclin D1 and D2 protein. Taken together, these

results demonstrate that ERβ mediates BPA-induced proliferation in adrenal cells.

The ability of estradiol to activate the shh signaling pathway has previously been

demonstrated in ERa positive breast and gastric cancer cells 45-47, however it remains

unknown if a similar effect can be observed through ERβ. Therefore, to determine if ERβ

activates the shh signaling pathway in adrenal cells, we examined the effects of ERβ

specific agonist DPN on expression of the two key proteins in the shh signaling pathway.

We found that DPN increased both shh and Gli1 protein levels, indicating a novel link

between ERβ and shh activation. We then determined if the activation of ERβ by BPA

leads to activation of the shh signaling pathway. We treated cells with BPA in the

presence and absence of ERβ-specific antagonist PHTPP, and found that PHTPP

abrogated the stimulatory effects of BPA on shh and Gli1 protein levels. Collectively,

these results indicate that BPA stimulates adrenal cell proliferation via ERβ-induced

activation of the shh signaling pathway.

In conclusion, the present study demonstrates for the first time that BPA acts on ERβ to

activate the shh signaling pathway, which in turn leads to increased proliferation in

H295A cells. Thus, our present study reveals a novel BPA-induced cell proliferation

signalling pathway that may underlie the increased adrenal gland weight phenotype we

reported previously in prenatally BPA exposed adult mouse offspring.

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44 Castillo, A. B., Triplett, J. W., Pavalko, F. M. & Turner, C. H. Estrogen receptor-β regulates mechanical signaling in primary osteoblasts. Am J Physiol Endocrinol Metab 306, E937-944, doi:10.1152/ajpendo.00458.2013 (2014).

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51 Ramos, J. G. et al. Bisphenol a induces both transient and permanent histofunctional alterations of the hypothalamic-pituitary-gonadal axis in prenatally exposed male rats. Endocrinology 144, 3206-3215, doi:10.1210/en.2002-0198 (2003).

52 García-Arévalo, M. et al. Maternal Exposure to Bisphenol-A During Pregnancy Increases Pancreatic β-Cell Growth During Early Life in Male Mice Offspring. Endocrinology 157, 4158-4171, doi:10.1210/en.2016-1390 (2016).

53 Brannick, K. E. et al. Prenatal exposure to low doses of bisphenol A increases pituitary proliferation and gonadotroph number in female mice offspring at birth. Biol Reprod 87, 82, doi:10.1095/biolreprod.112.100636 (2012).

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5 DISCUSSION AND CONCLUSION

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5.1 Summary This thesis utilizes both in vivo and in vitro approaches aimed at determining the effects

of prenatal exposure to environmentally relevant doses of BPA on adrenal gland

development and steroidogenic function. Prenatal exposure to BPA resulted in altered

adrenal steroidogenesis, as evidenced by increased plasma corticosterone levels.

However, there were no corresponding changes in plasma levels of ACTH. The increased

plasma corticosterone in female offspring was likely a result of enhanced adrenal

expression of StAR, the rate limiting factor of steroidogenesis. However, adrenal StAR

expression was not altered in male offspring, suggesting that BPA exerted sex-specific

effects on adrenal StAR expression. Using the H295A cell line as an in vitro model

system, I demonstrated that BPA induced StAR protein expression through an ERa-

and/or ERb-mediated unknown mechanism that was independent of transcription,

translation, and protein half-life. I also provided evidence that BPA increased adrenal

cortical cell proliferation in vitro via a novel molecular mechanism that involves ERb-

mediated activation of the Shh signaling pathway. Taken together, these findings

demonstrate that prenatal BPA exposure at environmentally relevant doses disrupts

adrenal gland development and steroidogenic function specifically pertaining to

glucocorticoid production. They also suggest that BPA-induced aberrant adrenal gland

development and steroidogenic function help explain alterations in plasma glucocorticoid

levels and HPA dysfunction seen in epidemiological 1,2 and experimental animal studies 3-6.

5.1.1 Doses and concentrations of BPA used in in vivo and in vitro experiments

The dose of BPA used in Chapter 2 (25 mg BPA/kg diet; equivalent to 5 mg BPA/kg

body weight) was chosen based on our previous prenatal exposure dose-response studies

in which impaired fetal lung, pancreas, and liver maturation were induced without any

effect on fetal body weight or litter size 7-9. This dose is also one tenth of the LOAEL

reported for rodents (50 mg/kg/day), as determined by the U.S. Environmental Protection

Agency (IRIS 2012). Importantly, maternal plasma concentrations of BPA in our mouse

model were determined to be 1.7 ng/ml, measured using GC–MS 7. The concentration of

BPA (10 nM) used throughout Chapters 3 and 4 is equivalent to 2.28 ng/ml, which is

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consistent with previous in vitro studies 10. The doses and concentrations of BPA used in

both in vivo and in vitro experiments are at the lower end of the exposure range reported

in the plasma (0.5-22.3 ng/ml) 11 and urine (0.16-43.42ng/ml) 1,2 of pregnant women in

the North America. Additionally, pharmacokinetic studies have shown that oral

administration of unconjugated BPA resulted in similar plasma levels in rodents and non-

human primates 12. Thus, the dosages of BPA as well as the mouse model and the adrenal

cortical cell line used in this thesis are relevant to human exposure.

5.1.2 Prenatal BPA disrupts adrenal steroidogenesis in a sex-specific manner

Exposure to EDC during critical periods of organ maturation affect lifelong organ

structure and function, leading to disease and dysfunction later in life 13,14. Evidence

suggests that EDC exposure during fetal development can lead to HPA axis programming

alterations, resulting in HPA and adrenal dysfunction in adulthood 15,16. As such, prenatal

BPA exposure in humans is associated with HPA axis dysfunction in both pregnant

women and 3-month old infants 1,2. In addition, animal studies have shown that perinatal

BPA exposure caused HPA dysfunction in adult offspring, resulting in changed gene

expression in the hypothalamus and pituitary, and altered plasma levels of CRH, ACTH,

and glucocorticoid 3-6. Moreover, Panagiotidou, et al. 6 demonstrated that BPA exposure

during pregnancy and lactation resulted in increased adrenal gland weight and abnormal

adrenal cortex zone thickness. Taken together, these findings suggest that developmental

exposure to BPA can disrupt the HPA axis and adrenal gland development, leading to

altered adrenal function in adulthood.

In Chapter 2, the effects of developmental BPA exposure on adrenal function were

assessed by examining the hypothesis that prenatal exposure to environmentally relevant

doses of BPA disrupts adrenal gland development and steroidogenic function in adult

mouse offspring. This study demonstrated that prenatal BPA exposure led to increased

basal plasma corticosterone levels independent of ACTH levels in both male and female

adult offspring. However, protein levels of StAR and cyp11A1, the two rate limiting

factors of steroidogenesis, were only increased in female offspring adrenal glands,

demonstrating the sex-specific effects of BPA.

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These findings are supported by numerous studies demonstrating the adverse effects of

other environmental chemicals on adrenal steroidogenesis. As described in Table 5.1,

pesticides, fungicides and insecticides (atrazine, imazalil, prochloraz, lindane, and 3-

methylsulfonyl-2,2-bis(4-chlorophenyl)-1,1-dichloroethene (3-MeSO2-DDE)) 17-23,

phytoestrogens (diadzein and geinstein) 18,21,22, organophosphates (Diethylumbelliferyl

phosphate and dimethoate) 18,24,25, nicotine 26, cadmium 18, mercury 27, and nonlyphenol 28

have all been shown to alter adrenal steroidogenesis by disturbing steroidogenic enzymes

and the downstream production of steroid hormone levels.

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Table 5.1: Environmental chemicals that alter adrenal steroidogenesis.

Environmental chemical Structural/ functional impact Reference

Atrazine Cyp19 inducer 17

Imazalil

Inhibit steroidogenic enzymes

Decreased cortisol and aldosterone

secretion

19

21,22

Prochloraz Inhibit cortisol and aldosterone

secretion through inhibition of cyp17 18

Lindane Increased steroidogenic enzymes and

StAR promotor activity 20

3-MeSO2-DDE Inhibit cortisol secretion through

interaction with cyp11B1 18,23

Diadzein

Inhibit activity but not expression of

3b-HSD

Decreased cortisol and aldosterone

secretion

21,22

18

Geinstein Inhibit activity but not expression of

3b-HSD

21,22

Diethylumbelliferyl phosphate Decreased cortisol and aldosterone

secretion 18

Dimethoate

Decreased cortisol and aldosterone

secretion

Inhibited StAR transcription

18

25

Nicotine Inhibit transcription of StAR 26

Cadmium Decreased cortisol and aldosterone

secretion 18

Mercury Inhibit testosterone and progesterone

levels 27

4-nonylphenol Decreased progesterone and increased

testosterone and 17b-estradiol levels 28

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Importantly, evidence suggests that dysfunction of the HPA axis during fetal

development results in increased glucocorticoid levels in adults, leading to an increased

risk for depression and other mood disorders 29,30. Thus, these studies provide a possible

mechanism behind BPA’s association with neurological and behavioural disorders such

as depression and anxiety 5,31.

Feedback and signaling pathways are essential for maintaining proper adrenal gland

development and function, and thus any alterations in these pathways may lead to disease

and dysfunction 32-35. Furthermore, there are a variety of transcription factors and

receptors that are essential for proper developmental processes and can be altered during

the critical period of organogenesis 32,33. Previous experimental animal studies have

provided evidence for an important role of the following factors and signaling pathways

in adrenal development: (1) steroid hormone receptors such as ER 36 and glucocorticoid

receptors 33; (2) Shh 37 and IGF signaling 38; and (3) transcription factors, such as SF-1 39

and DAX-1 40. Alterations in any of these factors result in abnormal adrenal development

and can potentially lead to altered adrenal function in adult life. Therefore, it is likely that

prenatal BPA disrupts fetal adrenal development by altering one or more of these factors

and signaling pathways.

5.1.3 BPA stimulates StAR protein expression through estrogen receptor signaling

In Chapter 2, I demonstrated that prenatal BPA exposure increased expression of the rate-

limiting factor of adrenal steroidogenesis, StAR, in adult female mouse offspring 41.

Therefore, H295A cells, the best available model of human fetal adrenal cortical cells,

were used an in vitro model system to investigate the underlying molecular mechanisms.

As shown in Chapter 3, BPA treatment increased StAR protein levels in a concentration-

dependent manner, indicating that these cells were a suitable in vitro model system.

Chronic induction of the rate-limiting step of adrenal steroidogenesis, StAR, suggests a

permanent upregulation of the steroidogenic pathway. This would result in an increase in

the production of glucocorticoids, which may lead to long term diseases such as

depression, anxiety, metabolic dysfunction, and glucocorticoid resistance. Additionally,

this sustained increase in adrenal steroidogenesis would suggest that BPA may disrupt

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HPA axis function. This is consistent with previous literature suggesting BPA affects the

HPA axis at the level of the hypothalamus and pituitary gland 3,5.

Since the ER pathway has been shown to be critical for adrenal gland development,

specifically for cortical development and ACTH receptor sensitivity 42, the possible

involvement of ER signaling was examined with BPA treatment. The ERa and ERb

isoform specific agonists PPT and DPN, respectively, were used to determine if ERa

and/or ERb were involved in regulating StAR expression. I demonstrated that both PPT

and DPN mimicked while the ER antagonist ICI blocked the stimulatory effects of BPA

on StAR protein levels. These findings suggested that the BPA-induced StAR protein

expression is likely mediated by ERa and/or ERb. They also revealed a novel role of

these two nuclear ERs in regulating adrenal StAR expression, and consequently adrenal

steroidogenesis.

I then elucidated the precise molecular mechanism by which BPA, via ERs, induces

StAR protein expression. First, I examined if BPA could be interfering with the

transcription of StAR mRNA, and found that levels of StAR mRNA were unchanged

after BPA treatment. This suggested that BPA did not affect StAR gene transcription.

Previous studies have found that StAR protein levels can be altered independent of

changes in StAR mRNA after exposure to oxysterols 43, pesticides 44, endotoxins 45 and

prostaglandins 46. However, the post-transcriptional mechanisms behind these effects

were not examined.

Next, I examined if BPA altered the translation of the 37-kDa StAR pre-protein, which

was found to be unchanged after BPA exposure, suggesting that BPA did not alter StAR

translation. The StAR pre-protein is cleaved into the mature 30-kDa isoform at the OMM

and transported to the IMM following cholesterol transport 47,48. If BPA interfered in the

processes of StAR cleavage or transport, there would not only be a change in the mature

StAR protein levels but also a reciprocal change in the 37-kDa pre-protein. Therefore,

since there was no change in StAR pre-protein levels, it is unlikely that BPA affected

StAR cleavage or transport.

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Lastly, I determined if BPA affected the half-life of StAR protein, and found that it was

unchanged following BPA treatment. An increase in mature StAR protein build-up in the

mitochondria is normally associated with an increase in corresponding proteases (LON

and AFG3L2) responsible for StAR degradation to prevent a novel form of mitochondrial

stress, termed StAR overload response (SOR) 49,50. Thus, the BPA-induced increase in

StAR protein levels independent of alterations in the half-life of StAR protein suggests

that BPA may alter the SOR. This buildup of StAR protein in the mitochondria may lead

to alterations in mitochondria structure and function 49.

Collectively, these findings suggest that BPA increases StAR protein levels likely

through ERa and/or ERb in adrenal cortical cells that involve an unknown novel

mechanism independent of StAR gene transcription, translation, and protein half-life.

Nevertheless, BPA-induced increases in StAR protein suggest the induction of adrenal

steroidogenesis, and consequently an increase in glucocorticoid production. However,

future research is required to decipher the precise molecular mechanisms behind these

effects to help better understand the adverse effects of BPA, as well as other EDCs, on

adrenal gland steroidogenesis.

5.1.4 BPA stimulates adrenal cortical cell proliferation through ERb-mediated activation of the Shh pathway

In Chapter 2, I demonstrated that prenatal BPA exposure increased both absolute and

relative adrenal gland weight without a change in plasma ACTH levels in adult mouse

offspring 41. Increased adrenal weight is normally a result of high plasma ACTH levels 51.

However, I did not observe an increase in basal plasma levels of ACTH, and concluded

that BPA likely affects adrenal gland weight independent of ACTH in our prenatal BPA

exposed mouse model 41. Therefore, I addressed this possibility in Chapter 4 using the

H295A cell line as an in vitro model system. Proliferation in the adrenal gland is essential

for development and remodeling in the adult 35,52. I demonstrated that BPA treatment of

H295A cells induced proliferation, as evidenced by increased levels of PCNA (a

universal proliferation marker), and key proliferation factors cyclins D1 and D2.

The Shh signaling pathway is heavily involved in the development and formation of the

adrenal glands, and Shh knockout mice display a severe hypotrophic adrenal gland

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phenotype 37,53. Furthermore, Shh signaling has been shown to induce proliferation in

other cell types, by increasing transcription of cyclin D1 and D2 genes 54. Analysis of the

Shh signaling pathway showed that BPA increased mRNA and protein levels of Shh, as

well as protein levels and activity (determined by nuclear translocation) of Gli1, a key

transcription factor in the Shh signaling pathway. Additionally, inhibition of the Shh

pathway by cyclopamine, a well-known Shh signaling pathway antagonist, blocked BPA-

induced increase in proliferation. Collectively, these results suggest that BPA activates

Shh signaling to increase adrenal cortical cell proliferation. Shh signaling pathway is not

only important for cell proliferation but is involved in a variety of other developmental

processes that may be altered due to BPA exposure 54. Previous studies have

demonstrated altered Shh levels in adrenal tumors in both adult and pediatric patients,

indicating its involvement in adrenal cancer, and suggesting that BPA may increase the

risk of adrenal carcinoma tumors 55,56.

The ability of estrogen to activate Shh signaling has previously been shown in breast and

gastric cancer cells 57-59. Therefore, I examined the hypothesis that BPA increases adrenal

cell proliferation through ERb-mediated activation of the Shh pathway. As shown in

Chapter 4, the ERb-specific antagonist PHTPP blocked the stimulatory effects of BPA on

PCNA, cyclin D1, and cyclin D2 levels, suggesting that ERb is involved in mediating

BPA-induced adrenal cell proliferation. Furthermore, PHTPP was also shown to prevent

the stimulatory effects of BPA on Shh and Gli1 protein levels, linking ERb to the

activating the Shh signaling pathway following BPA treatment. This is consistent with

previously reported findings that estrogen activates the Shh signaling pathway through

ERa 57-59. However, my findings demonstrate for the first time that ERb, through a direct

or indirect mechanism, regulates the Shh signaling pathway.

Taken together, these findings suggest that BPA acting through ERb activates the Shh

signaling pathway and results in increased adrenal cortical cell proliferation (Figure 5.1).

Changes and dysregulation in cell proliferation have previously been associated with

increased risk of cancer in numerous tissues 35,54,60. As well, BPA has been shown to be

associated with various types of cancers in both human and experimental animal studies 61-63. Therefore, it is tempting to speculate that an increase in adrenal cell proliferation

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could lead to an increased risk of adrenal cortical cancer. Moreover, higher levels of

adrenal proliferation in vitro suggest an increased potential for steroid hormone

production. If these findings could be extrapolated to humans, it is conceivable that

prenatal BPA exposure could lead to chronically elevated glucocorticoid levels. In

addition, due to the ubiquitous nature of both Shh and ER expression, there is the

potential that this novel BPA signaling pathway induced cell proliferation may be

applicable to other tissues, leading to numerous risks, including an increased risk of

cancer.

170

Figure 5.1: A schematic representation of the postulated molecular pathway by

which BPA stimulates adrenal cortical cell proliferation.

BPA readily crosses the cell membrane into the cytoplasm where it binds to and activates

ERβ. The activated ERβ translocates to the nucleus where it promotes transcription of the

shh gene, leading to increased shh mRNA and protein. Shh is secreted, acts in an

autocrine/paracrine fashion and binds to Ptch1 receptor, preventing Ptch1 from inhibiting

SMO. SMO is then released from the plasma membrane into the cytoplasm, leading to

the release of a complex containing the key shh transcription factor Gli1. Gli1

translocates to the nucleus where it binds to the promoters of key proliferation factors,

such as CCND1 and CCND2, and enhances their transcription, and ultimately leading

to increased cell proliferation.

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5.2 Future Directions The findings reported in this thesis reveal an important role of prenatal BPA in altering

adrenal gland development and steroidogenic function in adult mouse offspring, and

defines novel mechanisms of BPA actions in stimulating both adrenal StAR expression

and adrenal cortical cell proliferation. However, it is important to recognize there are

numerous important questions that need to be addressed, and are discussed below.

5.2.1 To study the effects of prenatal BPA on the other components of the steroidogenic pathway

The ZG is essential for the production of the steroid hormone aldosterone, which acts on

the distal tubule and collecting duct of the kidney to promote sodium reabsorption 64-66.

Aldosterone is synthesized from corticosterone by the enzyme Cyp11B2, localized in the

ZG, as part of adrenal steroidogenesis (Figure 1.8) 64-66. The regulation of aldosterone

production is not only controlled by ACTH, but is also regulated by angiotensin II and

potassium levels 64,65. Moreover, changes in aldosterone levels can result in altered blood

pressure due to its actions on sodium reabsorption 64,65. Indeed, epidemiological evidence

suggests an association between high BPA levels in plasma/urine and hypertension 67,68.

However, there are currently no known studies that have examined the effects of BPA on

aldosterone or Cyp11B2 levels. Thus, it is of importance to determine the potential

effects of BPA on aldosterone production and regulation, and to examine the

physiological consequences these effects may have.

The adrenal glands are also essential in producing precursors to sex hormones, such as

DHEA from the ZR 64,66. DHEA is not only required for the synthesis of estrogens and

testosterone in the adult reproductive system, but is critical for estrogen production by the

placenta during fetal development 64,66. Previous studies using an adult adrenal gland cell

line (H295R) have demonstrated that BPA treatment resulted in altered levels of

progesterone, testosterone, estrone, and estradiol 69. However, the effects of prenatal

BPA on DHEA levels in the adrenal glands and the potential mechanism behind these

effects are largely unknown. Therefore, further investigation into the effects and

mechanism of BPA’s actions on steroid hormones other than glucocorticoids in the fetal

and adult adrenal gland, and the long-term consequences of these effects, is warranted.

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5.2.2 To determine the precise molecular mechanism behind the effects of BPA on StAR protein levels

In Chapter 3, I was not able to completely elucidate the potential molecular mechanism

underlying the effect of BPA on StAR protein levels. However, I suggest that BPA does

not affect the gene transcription, translation, or protein half-life of StAR. Thus, further

research is warranted to examine the effects of BPA on StAR regulation in human

adrenocortical cells due to its essential role as the rate-limiting step in adrenal

steroidogenesis 47,70. The mechanisms underlying StAR regulation are not yet fully

understood, and new mechanisms and pathways are being continually discovered 47,71. As

such, BPA may affect StAR protein levels through an undiscovered novel mechanism.

However, there are a few possible mechanisms that may underlie BPA-induced StAR

protein levels, which include: (1) Changes in the 32-kDa isoform of StAR 72,73, which I was

unable to detect by western blotting. The role of the 32-kDa isoform of StAR is not fully

understood, yet it is interesting to speculate that an increase in the mature 30-kDa StAR

may be complimented by a corresponding decrease in the 32-kDa StAR; (2) alterations in

transport and/or cleavage of StAR protein at the OMM. It was proposed that a change in

transport and/or cleavage of StAR would need to be accompanied with a change in StAR

pre-protein. However, this may not be the case and further investigation to confirm this is

needed; and (3) changes in multiple steps of the StAR regulation pathway. As such, if

there are changes in multiple steps of the StAR regulation pathway, smaller changes in

individual steps may not be sensitive enough to be detected in these experiments. Further

studies need to examine each of these potential mechanisms.

5.2.3 To determine whether aspects of the signaling pathway identified in vitro can be observed in BPA-exposed mouse adrenal glands

In Chapter 4, I presented a novel molecular pathway by which BPA acts on ERb to

activate the Shh signaling pathway, which in turn leads to increased cell proliferation in

H295A cells. However, future studies are necessary to determine whether this same

molecular pathway operates in vivo in the adrenal glands of both fetal and adult mouse

offspring following prenatal BPA exposure. These include (1) measuring key

proliferation markers and factors (PCNA, cyclin D1, and cyclin D2); (2) determining the

173

levels (mRNA and protein) of key Shh signaling pathway factors Shh and Gli1, and their

activity in vivo (nuclear translocation of Gli1); and (3) examining levels of ERb and its

activity (nuclear translocation) in adrenal glands of both fetal and adult offspring after

prenatal BPA exposure. The foregoing experiments will ascertain whether the observed

mechanism in vitro is also in operation in vivo following prenatal exposure to BPA.

5.2.4 To determine the adrenal phenotype in ERa and ERb null mice

It has previously been shown that estrogen is essential for adrenal gland development in

rhesus monkeys 36,42,74. However, there are currently no known studies examining the

effects on adrenal gland development and/or function in ERa and ERb null mice.

Additionally, experiments in this thesis have demonstrated an important role of ER in

regulating adrenal steroidogenesis, as well as adrenal cortical cell proliferation via ERb-

mediated activation of the Shh signaling pathway. Thus, it is of important to characterize

adrenal gland phenotypes in both fetuses and adults of adrenal-specific ERa and ERb

knockout mice. These novel in vivo mouse models will define the role of ERa and ERb

during the development of the adrenal gland in the fetus, and the potential long-term

function of ERa and ERb in the adult offspring.

Generation of these mouse models would be useful in the future to further examine the

effects of BPA on adrenal gland development and function. Specifically, it will help to

further confirm the role of BPA in regulating adrenal steroidogenesis, and provide

definitive evidence to either support or refute if these effects are mediated through ERa

and/or ERb. These genetically modified mouse models will also be valuable in

confirming the role of ERb in activating the Shh signaling pathway, and consequent

stimulation of adrenal cortical cell proliferation. Additionally, these mouse models will

be invaluable in determining the mechanism of actions of other EDCs, acting through

ER, on adrenal gland development and function.

174

5.2.5 To determine the effects of BPA analogues on adrenal gland development and function

The continued research into the adverse effects of BPA has led to many manufacturers

discontinuing the use of BPA in their products 75,76. As such, BPA analogues such as

Bisphenol S, F, and AF (BPS, BPF, and BPAF) are being used increasingly as

replacement for BPA in products due to their similar chemical properties (Figure 5.2) 75,76. However, due to the structural similarities, there is potential for these chemicals to

not only exert the same effects as BPA but to cause other adverse effects. Indeed, recent

studies have found that exposure to BPS, BPF, and BPAF affect the reproductive 77,78,

endocrine 77, neurological 79,80, cardiovascular 81, and metabolic 82,83 systems.

It has previously been demonstrated that treatment of the H295R cell line (adult adrenal

gland) with increasing concentrations of BPS, BPF, and BPAF resulted in various effects

on steroidogenic function differing from that of BPA 84. Therefore, future in vitro and in

vivo studies are needed to further understand the potential adverse effects of these

bisphenol analogues on adrenal development and function, which will contribute to the

growing literature concerning the question of whether bisphenol analogues are safer

alternatives to BPA.

175

Figure 5.2: BPA and its analogues structure 84

176

5.3 Conclusions Insults during the critical period of development can often lead to long-term adverse

effects, resulting in disease and dysfunction in later life 13,14. Exposure to EDCs such as

BPA during critical periods of organ development is alarming for numerous reasons.

First, the presence of BPA in the environment is ubiquitous, and is detectable in the urine

of over 91% of Canadians 85. Second, BPA has been demonstrated to cross the placenta

and reach fetal circulation during organ development 86,87. Lastly, BPA is known to work

through several receptors and alter numerous cell signaling pathways that are involved in

organ development 61,88. While more attention has been placed on restricting/eliminating

the exposure of infants to BPA, specifically by banning BPA in baby bottles and

products, this does not prevent exposure of the fetus to BPA through maternal exposure 89,90. Epidemiological and animal studies have demonstrated the potential for prenatal

BPA to cause HPA dysfunction 1-6. However, the specific effects of prenatal BPA on the

adrenal gland development and function in later life remain largely unknown.

The experiments presented in Chapter 2 demonstrates the effects of environmentally

relevant doses of prenatal BPA induces adrenal steroidogenesis independent of ACTH

levels in a sex-specific manner in the mouse model. The next set of experiments,

described in Chapter 3, present a potential mechanism by which BPA induces StAR

protein expression, the rate-limiting factor of steroidogenesis, seen in adult female mice

prenatally exposed to BPA. Lastly, experiments in Chapter 4 provided evidence of BPA

altering adrenal development by increasing adrenal cell proliferation via ERb-mediated

activation of the shh signaling pathway in human adrenocortical cells. A schematic of this

molecular pathway through which BPA induces adrenal cell proliferation is shown in

Figure 5.1. This model speculates that BPA freely crosses the cell membrane, which then

binds to and activates ERb, leading to ERb translocation to the nucleus and consequently

increasing Shh mRNA and protein levels. Shh is then secreted and acts in an

autocrine/paracrine fashion to bind to Ptch1 receptor, prevent Ptch1 from inhibiting

SMO. SMO is released from the plasma membrane into the cytoplasm, leading to the

release of a complex containing Gli1, a key Shh transcription factor. Gli1 translocates to

the nucleus where it binds to the promoters of key proliferation factors CCND1 and

CCND2, and enhances their transcription and ultimately leading to increased cell

177

proliferation. This is a postulated model and further experiments are required to confirm

if this model translates into an in vivo model system. The long-term consequences of

increased cell proliferation in fetal adrenal glands must also be considered in the

application of this model.

Thus, this thesis supports the hypothesis that prenatal BPA exposure disrupts adrenal

gland development and steroidogenic function in adult offspring. These findings raise

concerns about the potential for adverse effects of prenatal BPA on adrenal gland

development and function, adding to the growing epidemiological evidence suggesting

adverse effects of BPA on the HPA axis and adrenal gland, and which we hope will

promote the continual banning of BPA in consumer products by regulatory agencies.

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Appendices

Appendix 1

187

Curriculum Vitae

Samantha Medwid

EDUCATIONAL BACKGROUND

PhD Physiology and Pharmacology

September 2013–December 2017

Western University, London, Ontario, Canada

Department of Physiology and Pharmacology

Thesis: EffectsofprenatalbisphenolAonadrenalglanddevelopmentand

steroidogenicfunction

Hon. BSc Nutritional and Nutraceutical Sciences

September 2009–June 2013

University of Guelph, Guelph, Ontario, Canada

Department of Human Health and Nutritional Sciences

SCHOLARSHIPS AND AWARDS

George W. Stavraky Teaching Award 2017

Obstetrics & Gynecology Graduate Scholarship (OGGS) 2015-2016

Western Graduate Research Scholarship 2013-2017

Nomination for Graduate Student Teaching Assistant Award 2015-2016 & 2016-2017

PUBLICATIONS

MedwidS,GuanH,YangK(2017).PrenatalexposuretobisphenolAdisruptsadrenal

steroidogenesisinadultmouseoffspring.EnvironmentalToxicologyandPharmacology.

43:203-208

Medwid S, Guan H, Yang K (2017) Bisphenol A induces steroidogenic acute regulatory

protein (StAR) expression via an unknown mechanism independent of transcription,

translation and protein half-life in human adrenal cortical cells. Submitted Steroids.

188

MedwidS,GuanH,YangK(2017).BisphenolAstimulatesadrenalcellproliferationthrough

ERβ-mediatedactivationofthesonichedgehogsignalingpathway.SubmittedJournalof

SteroidBiochemistryandMolecularBiology.

WestermanC,MedwidS,GuanH,YangK(2017).EffectsofprenatalbisphenolAexposure

ontheexpressionofwaterhomeostasisgenesinkidneysofadultmouseoffspring.In

preparation.

AbouTakaM,MedwidS,GuanH,YangK(2017).PrenatalbisphenolAcausessex-specific

effectsonliverfunctioninadultmouseoffspring.Inpreparation.

ABSTRACTS

Medwid S, Guan H, and Yang K. Bisphenol A stimulates adrenal cell proliferation

through ERb-mediated activation of the sonic hedgehog signaling pathway. 15th Annual

Paul Harding Research Day (1st place oral presentation), London Ontario, May 2017

Medwid S, Guan H, and Yang K. Bisphenol A stimulates adrenal cell proliferation

through ERb-mediated activation of the sonic hedgehog signaling pathway. Southern

Ontario Reproductive Biology Day (Poster Presentation), London Ontario, May 2017

Medwid S, Guan H, and Yang K. Bisphenol A stimulates adrenal cell proliferation

through ERb-mediated activation of the sonic hedgehog signaling pathway. London

Health Research Day (Poster presentation), London Ontario, March 2017

Medwid S, Guan H, and Yang K. Prenatal exposure to bisphenol A disrupts adrenal

steroidogenesis in adult mouse offspring. 14th Annual Paul Harding Research Day (2nd

place poster presentation), London Ontario, May 2016

Medwid S, Guan H, and Yang K. Prenatal exposure to Bisphenol A disrupts adrenal

steroidogenesis in adult mouse offspring. London Health Research Day (Poster

presentation), London Ontario, March 2016

Medwid S, Guan H, and Yang K. Prenatal exposure to Bisphenol A disrupts adrenal

189

steroidogenesis in adult mouse offspring. 13th Annual Paul Harding Research Day (Oral

presentation), London Ontario, May 2015

Medwid S, Guan H, and Yang K. Effects of prenatal Bisphenol A exposure on fetal

adrenal gland development in the mouse. Developmental Biology Day (Poster

presentation), London Ontario, June 2015

Medwid S, Guan H, and Yang K. Effects of prenatal Bisphenol A exposure on fetal

adrenal gland development in the mouse. 12th Annual Paul Harding Research Day

(Poster presentation), London Ontario, May 2015

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